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APL RSC U APLRUN PRG UTRNATHRT1 s{|STAR AWS |sTRNATHRT2 lz_PPP@$@ N Desk File Edit Break Options About APL ...-------------------- Desk Accessory 1 Desk Accessory 2 Desk Accessory 3 Desk Accessory 4 Desk Accessory 5 Desk Accessory 6 Save as ... )SAVE Open ... )LOAD Merge ... )COPY Delete ... )DROP--------------------- New )CLEAR Quit )OFF --------------------- Functions )FNS Variables )VARS Cut Copy Paste Clear Enter ----------- Open fn Close fn Quit fn Break Pause Resume Safety OPEN WHICH WORKSPACE ?SAVE WORKSPACE AS WHICH FILE ?NormalExpertA warning is issued afteran OPEN or CLEAR menu isselected, to prevent thecurrent workspace being lostNo warning is given beforeclearing or loading a newworkspace.Note : Commands from the keyboardwill not issue warnings.OKCANCELDELETE WHICH WORKSPACE ?MERGE WHICH WORKSPACE ?OKA Programming LanguageAtari ST Version 1.0 23/8/86Copyright (c) MicroAPL Ltd 1986APL.68000 _______________________________XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXEdit which function ?CANCELOK[1][Please make sure that you have|saved your workspace before|quitting. If not then cancel|now.][CANCEL|OK][1][Opening a new file now|will overwrite your current|workspace. Is this what you|want ?][CANCEL|OK][1][You are about to clear your|workspace. Please make sure|you have saved it before|proceeding.][CANCEL|OK][1][You are about to quit|from the function editor.|Is this what you want ?][CANCEL|OK] x :[! !PP" $ + 2  8 @  +P  J Z o  )=Sh ~'&    ! 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I &     5  6      / 6   6    6 ! ! N A3+E* A .&    E  Q   /%0"""$n$%R&&'6RES_SHIP.RSC/N/V/_/e/k/o/t/y//// Hello I am your Reset Ship Accessory Free Memory: Version 2.33 Set Mouse Speed: Settime Normal Warm ResetShow MouseProportionalCold ResetHide MouseDouble Prop.Ship HD'sAll Cancel Triple Prop. (c) Plaspoppie 1989 Turn off your Computer and Harddisk(s) now~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ It's quite useless to try a reset !! ______ Time: __:__:__ 999999________ Date: __/__\____ 99999999OKCancelJanuaryFebruaryMarchAprilMayJuneJulyAugustSeptemberOktoberNovemberDecember[1][ | Proportial mouse | not installed ][ OK ] RESET & SHIP [3][ | Warm Reset ?][ OK | NO ][3][ | Cold Reset ?][ OK | NO ][3][ | Ship HD('s) ?][ OK | NO ] %02d:%02d %2d %s %04d %6ld Bytes[2][ | Do you see your mouse now ?][ No | Yes ]%02d%02d%02d%02d%02d%04d%02d%02d%02d%02d%02d%04d>   )3>JWet 8d *z L * 86  "^2B.  , ~.X.Tj& ,$ ,  #a000000 #b000000 #c7770007000600070055200505552220770557075055507703111103 #d #E 18 13 #W 02 00 12 04 10 08 08 A:\STAR\*.*@ #W 00 00 0D 08 2A 0B 00 @ #W 00 00 0E 09 2A 0B 00 @ #W 00 00 0F 0A 2A 0B 00 @ #M 00 00 00 FF A FLOPPY DISK@ @ #M 00 01 00 FF B FLOPPY DISK@ @ #T 00 06 02 FF TRASH@ @ #F FF 04 @ *.*@ #D FF 01 @ *.*@ #G 03 FF *.APP@ @ #G 03 FF *.PRG@ @ #F 03 04 *.TOS@ @ #P 03 04 *.TTP@ @ #G 03 04 APLRUN.PRG@ *.AWS@  ________________ Acknowledgements STAR is developed by: (c) 1990 F.H.D. van Batenburg 2) J.P. Abrahams 1),2) M. van den Berg 2) V.W.A. Bos 2) C.W.A. Pleij 1) 1) Department of Biochemistry, Gorlaeus Laboratories, University of Leiden, the Netherlands. 2) Institute of Theoretical Biology, University of Leiden, the Netherlands. Every effort has been made to ensure the accuracy of this document, but no responsibility can be accepted for any errors or omissions It is understood that one copy of STAR will be used at a time. However, it may be used on more than one machine for educational purposes only and if the machines are at a single location (classroom). STAR is licensed on an as-is basis; no warranties or liabilities as to any incidental or consequential damages, loss of revenues, reputations, grants or data apply. Reselling or redistribution of STAR, or its parts, in another program system is forbidden. Apple and Macintosh are trademarks of Apple Computer Inc. Atari and ST are trademarks of Atari Corp. This software was developed using APL.68000, a proprietary product of MicroAPL Ltd, which has given permission for a runtime version of APL.68000 to be included with the software. Copyright and all intellectual property rights in APL.68000 remain vested in MicroAPL Ltd. APL.68000 is a trademark of MicroAPL Ltd. Version 1.1, june 1990 for STAR version 1.1. - 1 - Page 2 SSSSS SSSSS SSSSS I N S T A L L A T I O N G U I D E SSSSS SSSSS SSSSS S T A R SSSSS SSSSS SSSSS SSSSS f o r SSSSS SSSSS SSSSS SSSSS A t a r i S T SSSSS SSSSS SSSSS SSSSS a n d SSSSS SSSSS SSSSS SSSSS A p p l e M a c i n t o s h SSSSS SSSSS SSSSS SSSSS SSSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS - 2 - Installation Guide STAR Page 3 1 About this manual 29 June 1990 1 ABOUT THIS MANUAL In this manual you learn how to install STAR at your computer. In the first chapter we give the specifications of the hardware that STAR can use. In the next chapters we tell you how to install STAR on your Atari or Apple computer - 3 - Installation Guide STAR Page 4 2 Hardware Specifications 29 June 1990 2 HARDWARE SPECIFICATIONS STAR runs on Atari ST, Apple Macintosh and IBM PC-compatible computers. The IBM-PC version is slightly different from the Atari and Apple versions. Therefore we made a different manual for the IBM-PC version. In this manual we ignore this version. STAR supports: - Atari ST computers with a minimum memory of 1 Mbyte and at least one 720 Kb floppy disk drive. - Epson FX80 compatible matrix printers (Atari ST version) - Apple Macintosh computers. - Apple printers (Apple Macintosh version) - 4 - Installation Guide STAR Page 5 3 Installing STAR at an Atari ST 29 June 1990 3 INSTALLING STAR AT AN ATARI ST COMPUTER Before you do anything else, we strongly advise you to make a copy of the program disk we supplied with this package. If you don't know how to make a copy, consult the manual that was delivered with your computer. Afterwards you can store the original programdisk in a safe place and use the copy as your working disk. It is possible to move STAR to other disks, like a harddisk. This is a simple procedure. Just copu folder STAR from the floppy to your harddisk. Regardless of your configuration, you must always install APLRUN.PRG on your boot disk (floppy or hard disk). The procedure is as follows... 1. Open folder STAR. You do this by double clicking upon that folder. 2. Install 'APLRUN.PRG'. You do this by clicking once on the file 'APLRUN.PRG' and subsequent activation of the option [Install Application...] in the [Options] menu on the desktop. Type 'AWS' as the document type in the box. Activate the button [GEM]. Press the [OK] button. 3. Store this new setting. Activate option [Save Desktop] in the [Option]-menu. After this installation procedure, you can start the program by double click upon STAR.AWS. - 5 - Installation Guide STAR Page 6 4 Installing STAR at an Apple Macintosh 29 June 1990 4 INSTALLING STAR AT AN APPLE MACINTOSH COMPUTER Before you do anything else, we strongly advise you to make a copy of the program disk that we supplied with this package. If you don't know how to make a copy, consult the manual that was delivered with your computer. Then you can store the programdisk in a safe place and use the copy as your working disk. Before you try to run STAR, you must put the font that STAR uses in the system file of your startup diskette and restart. We placed the STAR font in the file called 'STARFONT'. You can put the STAR font in the systemfile using the Font/DA Mover that was supplied with your computer. Don't despair if you don't know how to use this program as we explain this step-by-step in the following paragraphs. In these paragraphs we don't include the switching of disks that might occur of you use a computer without a harddisk. 1. Activate the Font/DA Mover program. You do this by double-clicking the file with that name. 2. Put the Font/DA Mover in 'Font' mode. You do this by activating the [Font] button on top of the Font/DA Mover window. 3. Close the left window of the Font/DA Mover. You do this by pressing the [Close] button below the left window. 4. Open the STAR font in the left window of the Font/DA mover. You can do this by pressing the [OK] button below the left window and the [Option] key (or [ALT] key, depending on your keyboard) at the same time. Now a file-selector appears in order to open the folder called 'STARFONT'. If you don't know how to use the file-selector, please consult the manual which was delivered with your computer. 5. Open the systemfolder of your startup diskette in the right window of the Font/DA Mover. You do this by pressing the [Open] button below the right window of the Font/DA mover. Then a file-selector appears which enables you to open the system file. 6. Activate the file 'APL68000 10' in the left window of the Font/DA Mover. You do this by clicking once on the filename 'APL68000 10'. 7. Copy the STAR font to your system file. You do this by pressing the [>>Copy>>] button between the two windows of the Font/DA Mover. - 6 - Installation Guide STAR Page 7 4 Installing STAR at an Apple Macintosh 29 June 1990 8. Leave the Font/DA Mover. Just press the [Quit] button. We gave the STAR font number 210. If you use another font with that number, you should remove that font from your systemfile and put the STAR font 'APL68000 10' in it. In that case, you must restart before you use STAR. When you want to use that other font again, remove the STAR font and put the other font in the systemfile. If you use a font that is also named 'APL68000 10', you must remove it from your systemfile, place the STAR font in it and restart before you use STAR. If you want to use the original font again, remove the STAR font from the system file and place your font back in it. It is possible to move STAR to other disks, like a harddisk. This is a simple procedure. Just copy folder STAR from the floppy to your harddisk. Almost enough memory to the applications. The procedure is as follows. Click on APL.68000 once, followed by the [Get info] option of the [File]-menu. Next assign memory in the bottom-right box; the more the better but minimum 640K. After this installation procedure, you can start the program by double click upon STARAWS. - 7 - Page 8 29 June 1990 SSSSS SSSSS SSSSS T U T O R I A L SSSSS SSSSS SSSSS S T A R SSSSS SSSSS SSSSS SSSSS f o r SSSSS SSSSS SSSSS SSSSS A t a r i S T SSSSS SSSSS SSSSS SSSSS a n d SSSSS SSSSS SSSSS SSSSS A p p l e M a c i n t o s h SSSSS SSSSS SSSSS SSSSS SSSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS - 8 - CHAPTER 1 INTRODUCTION This tutorial introduces you to the capabilities of STAR and shows you how to use it to predict a secondary structure of a RNA molecule. Use this tutorial as an aid to learning, not as a detailed reference. For this, you find a Reference Guide elsewhere in your package. Before you read this tutorial, read paragraph 1.2 from the Reference Guide where you will find the conventions used. If you didn't install STAR yet at your computer, consult the Installation Guide. We advise you to read the chapters 2 to 4 of the Reference Guide also. These chapters give some background information about STAR and the folding of RNA and introduce you to the use STAR and its menu. It may also be useful to keep Appendix A from the Reference Guide ready while you are doing the exercises. This appendix illustrates the procedure of using STAR. - 9 - CHAPTER 2 ORGANIZATION OF THE TUTORIAL This tutorial contains explanations of the major features of STAR and exercises to get acquainted with STAR. It should take you between 2 and 3 hours to complete the tutorial in one session. Be sure to do the exercises in the presented order, since each exercise introduces concepts used in later ones. ________ _ Exercise 1 is a very quick example of how a RNA structure looks like in STAR. It also teaches you how to open RNA sequencefiles and structurefiles. ________ _ Exercise 2 teaches you how to view and edit a RNA sequence. ________ _ Exercise 3 teaches you how to save a RNA sequence. ________ _ Exercise 4 teaches you how to predict a secondary structure and how to view it. ________ _ Exercise 5 teaches you how to force double- and single-stranded regions into the structure which is to be predicted. ________ _ Exercise 6 teaches you how to view, change and save the energy rules that STAR uses for the calculation of a structure. - 10 - CHAPTER 3 EXERCISES 3.1 EXERCISE 1 You are probably eager to let STAR produce a structure on your screen. This exercise is a very quick example of the output that STAR produces. For this example, follow the steps shown below. First we open a sequencefile from disk. This file contains the ______ sequence of the tRNA thrT gene from E.Coli. 1. Activate the menuoption [Primary/Open]. a file-selector appears which you can use to open the file 'TRNATHRT.1' from the programdisk. If the file-selector shows the root, you should open folder STAR (double click upon this folder) in which file 'TRNATHRT.1' resides. If you don't know how to use a file-selector, consult the manual that was delivered with your computer. Now we open a structurefile from disk. This file contains ______ the structure of the thrT tRNA gene from E.Coli as predicted by STAR. 2. Activate the menuoption [Secondary/Open]. (*1) A file-selector appears which you can use to open the file 'TRNATHRT.2' (also in folder STAR) from the programdisk. Now we display the opened structure on screen. 3. Activate the menuoption [Secondary/View]. A box appears on the screen. Press [Span] followed by [OK]. Now STAR calculates a graph of the structure. ------------------------------------------------------------ (*1) For Atari-users only: be careful that you wait untill the mouse-bee has changed into an arrow, before you activate a pull-down menu. Otherwise you mouse may dissapear once and for all. - 11 - STAR Tutorial Page 3-2 3 Exercise 1 29 June 1990 After a while the following graph appears on the screen: ------------------------------------------------------------ | ---------------- ----------------- ------------ | | | | | | |-----| |--| |--| |----| |----| |----| GCUGAUAUAGCUCAGUUGGUAGAGCGCACCCUUGGUAAGGGUGAGGUCGGCAGUUCCAAU : : : : : : : : : : : : 123456789012345678901234567890123456789012345678901234567890 1 2 3 4 5 6 ------------- ----- | | | |---||------| CUGCCUAUCAGCACCA : : : 1234567890123456 7 This is a graphical representation of the stems of the structure ______ of tRNA thrT from E.Coli as it is calculated by STAR. The graph is explained in paragraph 7.7.2 from the reference Guide. OK, this was the first exercise. It was a quick example of how a RNA structure looks like in STAR. You also learned how to open sequence and structure files. - 12 - STAR Tutorial Page 3-3 4 Exercise 2 29 June 1990 3.2 EXERCISE 2 In this exercise you learn how to view and edit a RNA sequence. First we look at the sequence you opened in the previous example. 1. Activate the menuoption [Primary/View]. STAR displays the sequence as shown below on screen. 5 10 15 20 25 30 35 40 45 GCUGA UAUAG UUGGU AGAGC CGAGC CUUGG UAAGG GUGAG GUCGG 50 CAGUU CCAAU CUGCC UAUCA GCACC A 100 As you see, STAR divides the sequence into parts of 5 nucleotides. On each line STAR shows 50 nucleotides. The numbers start with 1 at the 5' part of the sequence, and increase until the 3' part of the sequence is reached. Now we will edit the sequence. 2. Activate the option [Primary/Edit]. STAR replaces the 'normal' menubar by the editor menubar. Furthermore, you see the sequence as described above. At the first nucleotide you see a blinking dash. This is the cursor which indicates the position where you can delete or add nucleotides. 3. Now we will replace the 60th nucleotide (the U) by a C. First we move the cursor to the 60th nucleotide. You can do this with the 'arrowkeys', or faster with the mouse. When the cursor is at nucleotide 60, press the key marked with 'C' together with the [Shift] key. You see the U change into a C. Note that the cursor moves automatically to the 61th nucleotide. 4. Now we will insert a new nucleotide (the G) between nucleotide 61 and 60. Your cursor is still at position 61. Press the [insert] key. You see the cursor change from a blinking high dash to a blinking low dash. This indicates that you are working in 'insert' mode. This means that you will insert the nucleotide you type in the sequence. This differs from the previous step which replaced the nucleotide at the cursor position. Press the key marked with 'G' together with the [Shift] key. - 13 - STAR Tutorial Page 3-4 4 Exercise 2 29 June 1990 STAR redisplays the sequence. You see that the G you typed is inserted so that nucleotide 55-65 are now: _ CCAAC GCUGC 5. Now we will delete the previously inserted nucleotide. Your cursor is at position 62. Press the [Backspace] key. Now STAR deletes the previously inserted G. Note that the cursor moves one position left to nucleotide 61. 6. We are not satisfied with the 60th nucleotide. This is the nucleotide we replaced at step 3. Therefore we will delete it. Move the cursor to the 60th nucleotide. Press the [Delete] key. Now STAR deletes the 60th nucleotide, the C. 7. Now we move the first 10 nucleotides to the end of the sequence. For this we use the clipboard. Move the cursor to the first nucleotide. Now we select nucleotide 1 to 10. Point your mouse at the first nucleotide and press the (for Atari left) mousebutton (the mousekey). _____ ___ ____ ________ Move the mouse to nucleotide 10 while you keep pressing ___ ___________ the mousebutton. Release the mousebutton. STAR shows nucleotide 1 to 10 in 'reversed video': white letters on a black background. This means that you can now move it to the clipboard. This method to select nucleotides is called 'dragging'. Activate the option [Clipboard/Cut]. STAR transfers the selected nucleotides to the clipboard. Now we paste the clipboard contents at the end of the sequence. Move the cursor to the end of the sequence. This is one position after the last nucleotide. Activate the option [Clipboard/Paste]. STAR places a copy of the clipboard contents (the 10 nucleotides previously selected) at the end of the sequence. 8. Now we duplicate the whole sequence. Again, we use the clipboard. Select the whole sequence using the method described above (dragging). Activate the option [Clipboard/Copy]. Now STAR places a copy of the sequence in the clipboard. Move the cursor to the end of the sequence. Activate the option [Clipboard/Paste]. The sequence is - 14 - STAR Tutorial Page 3-5 4 Exercise 2 29 June 1990 duplicated. 9. We want to change all C nucleotides into A nucleotides. Activate the option [File/Replace]. A box appears in which you can type two characters: the nucleotide that you want to replace and the nucleotide you want to replace it by. Type 'C' at the leftmost field and 'A' at the rightmost one. Press the [OK] button. STAR replaces all C nucleotides by A nucleotides. This feature is especially usefull for converting DNA sequences into RNA sequences; in that case you want to replace all T's by U's. 10. We want to delete all A nucleotides Again, we use option [File/Replace]. Now you type 'A' in the leftmost field and ' ' at the rightmost one. Press the [OK] button. Now STAR deletes all A nucleotides in the sequence. 11. Imagine you are tired of your sequence and you want to work with another one. Point your mouse at the first nucleotide and press the (for Atari left) mousebutton (the mousekey). _____ ___ ____ Move the mouse the last nucleotide while you keep ________ ___ ___________ pressing the mousebutton. Release the mousebutton. Now STAR shows all nucleotides in 'reversed video': white letters on a black background. This means that you can now move it to the clipboard. Activate the option [Clipboard/Cut]. STAR transfers the selected nucleotides to the clipboard. Type in a new sequence, for example: CACCCUG 12. Finally we leave the editor. Activate the option [File/Exit]. After a short while the 'normal' menubar reappears. If you now view the sequence (see point 1), you see that it is identical to the one in the editor. The sequence which existed before the editing is gone! This is because we used the [File/Exit] option. With this option you leave the editor and the sequence is trans- ferred from the editor to the main STAR menu. Another way to leave the editor is the [File/Quit] option. If you use this option, you also leave the editor but - 15 - STAR Tutorial Page 3-6 4 Exercise 2 29 June 1990 then STAR discards the sequence in the editor. So, this was the first 'real' exercise. In this exercise you learned how to view and edit a sequence. You learned how to use the keys and menuoptions during the editing. You learned two different ways to leave the editor. - 16 - STAR Tutorial Page 3-7 5 Exercise 3 29 June 1990 3.3 EXERCISE 3 In this exercise you learn how to save a sequencefile to disk, so that you can use it another time with STAR. 1. Activate the option [Primary/Save as...]. a file-selector appears which enables you to enter a filename. As the sequence we made in the previous exercise contains nothing intelligible, enter 'Rubbish.1' as the filename. Now STAR saves the sequence to that file. After saving, a bell sounds. 2. Now we will check whether we really saved the sequence. First we open a new sequence. Like in exercise 1 we will open 'TRNATHRT.1'. Activate the option [Primary/Open]. A warning appears: 'Opening a new sequence will overwrite your current sequence'. Since we have just saved our current sequence, press [OK]. a file-selector appears which enables you to select the file 'TRNATHRT.1' Check if we really opened a new sequence. Use the option [View] (see previous exercise). 3. Now we will open our 'old' sequence 'RUBBISH.1'. Again, use the option [Primary/Open]. If you examine the opened sequence ([Primary/View]) you see the sequence we produced in the previous example. Imagine that you are tired of working with a particular sequence and want to work with another one. In exercise 2 you learned how to do that by replacing the nucleotids by typing the new string yourself in the [Edit] after cutting the old nucleotids. If however the other string is already defined in a file (copied from a bulletin board, or sent to you by e-mail), you simply activate option [Primary/Open]. Next you read this string as you did in this exercise. This was a short exercise in which you learned how to save a sequence for later use and in which you learned how to read this (or any other) sequence. - 17 - STAR Tutorial Page 3-8 6 Exercise 4 29 June 1990 3.4 EXERCISE 4 In this exercise you will learn how to simulate the folding of a RNA molecule and how to examine the predicted structure. Furthermore you will learn how to stop using STAR. We want to start without a sequence and a structure. The easiest way to do this is to start STAR up again. Therefore we first stop STAR. 1. Activate the option [Files/Quit]. A box appears which asks you if you really want to stop using STAR. Press [OK], as this is what we want. Now you can start STAR up again. 2. Open the sequence 'TRNATHRT.1' from the program disk. Use the option [Primary/Open] which we explained in the first exercise. 3. Activate the option [Secondary/Calculate] The program will now simulate the folding of the RNA molecule. First STAR creates a stacking table. It displays the size of the submatrices it searches on the screen. Next STAR adds the most stable stems to the structure. It displays the added stems one by one on the screen. After a while (less than 3 minutes with Atari or MAC-plus), the calculation ends. STAR displays the predicted structure: 27 32 38 43 * -15.9 4.1 * CACCCU * GUGGGA 49 53 61 65 * -12.0 4.7 * GGCAG * CCGUC 1 7 66 72 * -14.4 4.6 * CGUGAUA * CGACUAU 11 13 22 22 * -4.2 5.0 * CUC * GAG - The first 4 columns define the stems of the structure: they represent the positions of the upper and lower nucleotides of the stem halves. For example: the first stem is formed by basepairing the nucleotides 27-32 with nucleotides 38-43. - The fifth column represents the energy contents of the stem in kcal/mol. - The sixth column represents the energy contents of the single-stranded RNA enclosed by the stem in kcal/mol. - 18 - STAR Tutorial Page 3-9 6 Exercise 4 29 June 1990 - The remaining columns represent the nucleotides present in the stem. This was the third exercise. In this exercise you learned how to use STAR to calculate (predict,fold) a secondary structure. - 19 - STAR Tutorial Page 3-10 7 Exercise 5 29 June 1990 3.5 EXERCISE 5 It is possible that you know that some or more stems exist in the secondary structure. In this exercise you learn how to force those stems in the structure that STAR calculates. Moreover, you could be sure that some parts of the sequence do not basepair. In this exercise you also learn how to force STAR to incorporate such single-stranded parts in the structure. Again, we start without a sequence and without a structure. The easiest way to do so is to stop STAR and start it up again. In the previous exercise you learned how to do this: 1. Open the sequence 'TRNATHRT.1' You can force STAR to incorporate certain stems in the structure by defining those stems before you start the calculation. 2. Activate the option [Secondary/Edit]. You now enter the editor for structures. This editor is basically similar to the one we used in exercise 2, except that you work with four columns. Those columns represent the top and bottom nucleotides of the stems in the structure. As we don't have a structure yet, you see a screen with four empty columns. 3. Now enter the stem '5 10 15 20'. Type '5', [TAB], '10', [TAB], '15', [TAB], '20'. Note that you should use the [TAB] key to move from column to column. This is one way to enter a stem. 4. Another way to enter a stem is to use the [File/Glue] option. Activate the option [File/Glue]. The sequence appears on screen the same way as described in exercise 2. Now you can use the mouse the point at the sequenceparts that pair to form a stem. We use this method to enter the stem '25 30 35 40' Point your mouse at nucleotide 25 and press the mousebutton. Move the mouse to nucleotide 30 while you keep pressing the mousebutton. STAR shows nucleotides 25-30 in reversed video: white letters on a black background. Next define the other stemhalf (35 40) in the same way. A box appears which asks if you want to add the stem you entered to the structure. If you made no mistakes, - 20 - STAR Tutorial Page 3-11 7 Exercise 5 29 June 1990 press [OK], otherwise press [Cancel]. A box appears which asks if you want to enter another stem (using the [Glue] option). If you made no mistakes, press [OK], else press [Cancel] and you get another chance to enter the stem. 5. Leave the editor. We explained how to do this in exercise 2. As we want to save the stems we entered, use the [File/Exit] option. 6. Check whether you really entered the two stems. Use the option [Secondary/View]. If you view by values you see the stems: 5 10 15 20 0.0 0.0 AUAUAG CGAGAU 25 30 35 40 0.0 0.0 CCUUGG GAGUGG You can force single stranded regions to occur in the predicted structure in the following way: change the nucleotides in the sequence into a character other then A, U, C or G. For clarity change the nucleotides to the same character in lowercase. 7. Choose the menu option [Primary/Edit] and change the nucleotides with number 11 and 12 (C and U) to lowercase. 8. Choose the menu option [Secondary/Calculate]. Now a box appears that warns you that STAR will add stems to the structure you just defined. We want to incorporate the defined structure in the predicted structure, so click on the box with [OK]. STAR will now simulate the folding of the RNA and incorporate the predefined stems. This will take some time (about 3 minutes for Atari and MAC-plus). When STAR is finished it displays the predicted structure. Note that the predefined stems and the single stranded region are incorporated in the predicted structure. The predefined stems are the occurring first in the structure, the energy of these stems and the single stranded region enclosed by these stems are not calculated, but set to 0. - 21 - STAR Tutorial Page 3-12 8 Exercise 6 29 June 1990 3.6 EXERCISE 6 In this exercise you learn how to view, edit and save the energy rules that STAR uses to calculate structures. If you are satisfied with the energy rules that are built in STAR as a default, and you don't want to experiment with them, you don't need to do this exercise. First we will view the energy rules. 1. Choose the option [Energyrules/View]. A box appears by which you can select one of the seven types of energy rules. These types are explained in paragraph 8.5 of the Reference Guide you find elsewhere in this package. 2. Select the 'Stacking' energy rules: press the [Stacking] followed by the [OK] button. STAR now displays the stacking energies it uses to predict structures. For the interpretation of these stacking energies, consult paragraph 8.5.1. of the Reference Guide. 3. Take a look at the energy rules for bulge loops, interior loops, hairpin loops, deep grooves and shallow grooves. Again, use the option [Energyrules/View]. For the interpretation of these energy rules, consult paragraphs 8.5.2-8.5.7. of the Reference Guide. We will now edit the stacking energy rules. 4. Choose the option [Energyrules/Edit]. Select the stacking energies as we explained at point 2. You now enter the editor. On your screen you see the stacking energies we explained at point 2. At the upper left corner of your screen, you see the blinking cursor you already know from exercise 2 and 5. 5. Move the cursor to the energy value of the stack GC-GU. Look in the right column by GU, in the top row by GC. The point where these lines cross, is the energy value for the stack GC-GU. The energy value for this stack is -1.3 kcal/mol. 6. Replace this value with 99. You can do this using the same keys as explained in exercise 2. As you change this value, notice that the energy value of the stem UG-CG also changes, because both energy values are equal. 7. Activate the [QUIT] option to leave the editor. The changes you made are NOT incorporated in the energy rules. - 22 - STAR Tutorial Page 3-13 8 Exercise 6 29 June 1990 We will now change the energy rules of interior loops. The editing of bulge loops and hairpin loops is similar. 8. Again, activate option [Energyrules/Edit] and select the energy rules for interior loops. A box appears which tells you that the interior loop energy rules are too big to fit in the editor. The maximum number of rows you can edit at a time is 18 (Atari users), or 21 (Apple users). Reply that you want to edit row 1 to 10 and press [OK]. 9. Now you see the first 10 rows of the interior loop energy rules. These are the energy values for loops of 2-12 nucleotides long. 10. Delete the energy rules for loops 5 nucleotides long. You can, of course, delete each value for this loop of length using either the [Delete] or the [Backspace] key. Unfortunately, to delete 5 values that way takes some time. It is easier to use the clipboard. As in exercise 2, you first select that part of the energy rules you want to put in the clipboard. In contrast to the sequence editor, you can only select row-by-row here; for example it is not possible to select a single value. So, move the cursor anywhere in the row with the energy rules for interior loops of length 5. This is the fourth row. Press the mousebutton and keep it pressed for about one second. During this pressing, don't move the mouse to another row because STAR will select that row too. Release the mousebutton. The row is now selected. 11. Use the [Clipboard/Cut] option to delete the row. 12. Leave the editor using the [File/Quit] option. Again, you will not save the changes you made during the edit session. Now we will edit the energy rules for shallow grooves. Editing of the energy rules for deep grooves and backbones is a similar procedure. 13. Again, choose the option [Energyrules/Edit]. Select the energy rules for deep grooves. Now you enter the editor. On your screen you see the energy rules for deep grooves, together with four 'extra' columns filled with 0. You can use these columns to add extra energy values. - 23 - STAR Tutorial Page 3-14 8 Exercise 6 29 June 1990 14. Now add a column with energy values for the case that 16 nucleotides cross the shallow groove. Move to one of the columns filled with 0 and type your energy values there. 15. Leave the editor using the [File/Quit] option. Again, you will not save the changes you made during the edit session. This was the final exercise. In this exercise you learned how to view, edit and save the energy rules that STAR uses to calculate RNA secondary structures. Now you should be able to use STAR at your own. If you meet any problems, consult the Reference Guide you find elsewhere in this package. - 24 - Page 3-15 29 June 1990 SSSSS SSSSS SSSSS R E F E R E N C E G U I D E SSSSS SSSSS SSSSS S T A R SSSSS SSSSS SSSSS SSSSS f o r SSSSS SSSSS SSSSS SSSSS A t a r i S T SSSSS SSSSS SSSSS SSSSS a n d SSSSS SSSSS SSSSS SSSSS A p p l e M a c i n t o s h SSSSS SSSSS SSSSS SSSSS SSSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSS SSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSSSSSSSSS SSSSSSSSSSS SSSSS SSSS SSS SSSS SSSSS SSSSS SSSSS SSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSSSSSSSSS SSSS SSSSS SSSSS SSSSS SSSS SSSSS SSSS SSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS - 25 - CHAPTER 1 ABOUT THIS MANUAL This chapter shows you the organization of this manual. Furthermore it introduces you to the conventions we use in this manual. Therefore it is absolutely essential to read this chapter before you read on. ___ ____________ __ ____ ______ 1.1 THE ORGANIZATION OF THIS MANUAL We divided this manual into three parts. First, the introductionary part consists of chapter 1 to 3. This gives some general information about STAR and its use. Second, the reference part extends from chapter 4 to 8. This part explains all menus of STAR in detail. Finally three appendices provides information that you need frequently while using STAR. Before you start to work with STAR, we advise you to read the chapters of the introductionary part. ___ You should not plunce directly into chapters 4 to 8. They are not ment to familiarize the reader with STAR. If you need to learn STAR, you better start with the tutorial. At the start of each chapter we present a brief introduction about its contents and its purpose. ___________ ____ __ ____ ______ 1.2 CONVENTIONS USED IN THIS MANUAL This manual uses several conventions. We list them in this paragraph. ___ When we talk about a 'key', we mean a key from the keyboard. ______ With a 'button' we mean either the mouse-button (-key) or a button in a box. An example of a button in a box is the [OK] button. - 26 - Reference Guide STAR Page 1-2 1 About this manual 29 June 1990 ____ _____ ___________ _______ We write keys, menus, menuoptions and buttons, like OK buttons, between []. For example, the [Files] menu and the [Backspace] key. _____ We write names of things such as folders between '', for example: 'foldername'. _______ We enclose options in a menu with []. We separate the menu and its option with /. For example the [Primary/Open] option. Most of the time we don't include the menuname. In that case we mean the option from the menu that we discuss in the current chapter. For example: we discuss the [Primary] menu in chapter 6. In that chapter we refer to the [Primary/Open] option as [Open]. ______ In order to pres a button, you should point the mouse-arrow to that button and press the mouse button. For example 'press the [OK] button' means that you should point your mousearrow to the [OK] button and press the mousebutton. Atari users should use the left mousebutton. ___ When we refer to a box, we mean the window that appears on the screen with information and buttons in it. Some boxes have only one button, for example an [OK] button. You can leave these boxes by pressing that button, but you can also press the [CR] key. This is the key marked with 'Enter', 'Return' or with a large hooked arrow. Some boxes have two buttons to leave the box, for example an [OK] and a [Cancel] button. In this case you can also leave the box by pressing the [CR] key. This is equal to selecting the leftmost button of the box. For example: pressing the [CR] key in the following box has the same effect as pressing the [Cancel] button. +--------------------+ | Pressing [CR] has | | | | the same effect as | | | | pressing [Cancel] | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------+ Finally we want to define two frequently used terms: sequence and structure. ________ With sequence we mean the nucleotide sequence of the RNA molecule, the primary structure of the RNA. _________ With structure we mean the secondary structure of the RNA (with some tertiary elements; see paragraph 2.3). - 27 - Reference Guide STAR Page 1-3 1 About this manual 29 June 1990 ______ __ ____ 1.3 NAMING OF KEYS When working with STAR you can use several keys of the keyboard. Therefore we will explain which key corresponds with a certain name in the following paragraphs. We discuss the keys separately for Apple and Atari users. Especially Apple Macintosh users should read their paragraph thoroughly as some keys are normally not used in Apple programs. _____ ____ 1.3.1 Atari Keys In this paragraph we list the names of the various keys that we use in this manual. _____________ Characterkeys: these are the keys which marked with a letter from the alphabet. __________ Numberkeys: these are of course the keys marked with a number. _________ Arrowkeys: these are the keys marked with arrows. ____ ____ [CR] key: this is the key marked with 'ENTER', 'RETURN' or a large hooked arrow. ______ ___ [Ctrl] key: this is the key marked with 'CONTROL' or 'Ctrl'. ___________ [Backspace]: this is the key marked with 'Backspace' or a large leftward pointing arrow. ________ ___ [Delete] key: this is the key marked with 'DEL' or 'DELETE'. ________ ___ [Escape] key: this is the key marked with 'ESC' or 'ESCAPE'. ________ ___ [Insert] key: this is the key marked with 'INS' or 'INSERT'. _____ ____ 1.3.2 Apple Keys In this paragraph we list the names of the various keys that we use in this manual. _____________ Characterkeys: are the keys which are marked with a letter from the alphabet. __________ Numberkeys: these are of course the keys marked with a number. _________ Arrowkeys: these are the keys marked with arrows. ____ ____ [CR] key: this is the key marked with 'ENTER', 'RETURN' or a - 28 - Reference Guide STAR Page 1-4 1 About this manual 29 June 1990 large hooked arrow. _________ ___ [Command] key: this is the key marked with the cloverleaf-like sign. ___________ [Backspace]: this is the key marked with 'Backspace' or a large leftward pointing arrow. _______ ___ [Clear] key: this is the key marked with 'CLEAR'. Furthermore STAR uses some keys that are generally not used by Apple users. Those keys are positioned on the socalled numeric keyboard. This is the little keyboard at the right side of the large keyboard. Press these keys together with the [Ctrl] key. In the figure below we show you the numeric keyboard and the two keys you use while working with STAR. +-----+-----+-----+-----+ | | | | | |clear| = | / | * | | | | | | +-----+-----+-----+-----+ | | | | | | 7 | 8 | 9 | - | | | | | | +-----+-----+-----+-----+ | | | | | | 4 | 5 | 6 | + | | | | | | +-----+-----+-----+-----+ | | | |Enter| | 1 | 2 | 3 | | | | | | | +-----------+-----+ | | | | | [Command] + | 0 | . | | || || | | +3----------A3----+-----+ | | [Insert] [Delete] - 29 - CHAPTER 2 THE THEORY UNDERLYING STAR This chapter explains the theory that is the basis of STAR. We advise you to read this chapter. Then you should be able to make full use of the capabilities of STAR. ____________ 2.1 INTRODUCTION The folding of a single stranded RNA molecule is, at least for a great part, determined by its nucleotide sequence. This is the primary structure of RNA. The formation of base pairs like A-U, G-C and G-U gives rise to specific structural motifs. Such motifs are stem regions and single stranded regions like hairpin, bulge, multibranched and interior loops. The ensemble of these structural elements in a planar presentation is the secondary structure of RNA. As soon as a set of free energy values for the various structural elements was available, attempts were made to predict the most stable secondary structure for a given RNA sequence. Several algorithms have been developed for this purpose and they have become an additional tool in the determination of the structure of RNA. STAR is one of them which has some unique features. - 30 - Reference Guide STAR Page 2-2 2 The theory underneath STAR 29 June 1990 _______ __ ___ 2.2 FOLDING OF RNA We consider the formation of a RNA secondary structure as a stepwise process. During this process, intermediate structures evolve into the native one by subsequent addition of stems. Of all possible stems, only a few can be incorporated into the structure. Those stems forming fastest and lasting longest are preferred. You can measure the distance between two stemhalves by counting the number of nucleotides in the loop which results upon the formation of a stem. This distance determines the free energy that is needed to bring the two stemhalves near to each other. This free energy is an important determinant of the rate of stem formation. +------+ | | | | loop +--+---+ || stem 5'-------++------+ 3' Once the two stemhalves are near to each other, the efficiency of nucleation (initial basepair formation) determines their speed of association. Once the stem is formed, it is stable by itself, or last long enough to be stabilized by the formation of another string. The free energy that is released by the formation of a stem determines the efficiency of nucleation and the stability of that stem. The rate of formation of a stem thus depends on two types of free energy. These are the energy uptake for closing the loop and the energy release because of the pairing and stacking of the bases. Energy values of various loops and stacks of basepairs have been published. Almost all RNA secondary structure programs use these energy values. It is not clear how these free energies should be used to calculate the rate of stem formation. We solved that problem by simply adding the free energy of closing a loop to that of stacking and pairing. As an option you can multiply the first with a weighting factor. We assume that the resulting value is more or less proportional to the rate of stem formation. Therefore STAR incorporates the stem with the lowest value in the nascent structure. - 31 - Reference Guide STAR Page 2-3 2 The theory underneath STAR 29 June 1990 ________ ____________ 2.3 TERTIARY INTERACTIONS STAR is capable of dealing with interactions which are formed upon basepairing of a loop with a single stranded stretch. Because the energy of stacking stems upon each other is important for the formation of the final structure, we enumerated all possible ways in which this stacking can be reached. You can see them in the figures below. In this figures the "|" represent the RNA sequence, and ":" the basepairings. +---+ +---+ | | | | ++:++ ++:++ |:| |:| +:| +--+ |:| +:| | +-+:| |:| | +-+:| ++:++ +--- |:| | | |:| +---+ -+:++ fork bulge loop ----------------------------------------------------------- +-------+ +----------+ +----------+ | | | 3' | | 5' | ------- ------3+ +----+@----+ +----------+ ::::::|:::::| :::::|:::::: ::::::|:::: +------------+ 5'------3-----+ +-----3-----+3' | | | | | | +------+ +-----+ +-----+ pseudo-knot 3' a-knot 5' a-knot Note the 3' and the 5' a-knot. These pseudoknots are purely hypothetical so far; their existence in nature has not yet been proven. In these two types of pseudoknots you can distinguish three different domains. First, the quasi-continuous helical region. Second, one single stranded stretch which doesn't cross any groove. And third, one single stranded stretch crossing the shallow groove in the 5'a-knot, or crossing the deep groove in the 3'a-knot. The energy values which the program assigns to these regions depend on: 1. the number of bases crossing a groove. 2. the number of base pairs to be crossed. 3. the type of groove to be crossed (deep, shallow or none). - 32 - Reference Guide STAR Page 2-4 2 The theory underneath STAR 29 June 1990 The default energy values that are built in the program for these structures are based on an analysis of experimentally proven pseudoknots. For instance, we found a pseudoknot which can only exist if the energy values for its connecting loops are 4.2 kcal/mol or less. We therefore introduced this value for all pseudoknots whose connecting loops are shorter than 15 nucleotides. We prohibit the formation of other pseudoknots by assigning a value of 99.9 kcal/mol to the singlestranded stretches crossing both grooves. - 33 - Reference Guide STAR Page 2-5 2 The theory underneath STAR 29 June 1990 ____ ________ ____________ 2.4 LONG DISTANCE INTERACTIONS No systematic experimental studies have been performed on RNA fragments with loops longer than seven nucleotides. Therefore we have estimated energy values for longer loops by extrapolation using the theory of Jacobson and Stockmayer (*1) The program uses these extrapolated energy values as a default. However it is possible for you to change them. ------------------------------------------------------------ (*1) Jackson & Stockmayer (1950): Chem.Phys.(18)1600-1606 - 34 - Reference Guide STAR Page 2-6 2 The theory underneath STAR 29 June 1990 ___ _________ 2.5 THE ALGORITHM Unlike most of the RNA folding programs, the algorithm used in STAR does not calculate the most stable structure for a given RNA sequence. Instead of this, it simulates the folding of the RNA molecule. Although a RNA structure can form rapidly in nature, it does not form instantaneously. Instead we envision a stepwise process. As we see it, the sequential formation of several intermediate structures gives rise to the final, native structure. __________ When a stem is formed, the initiation of its formation is rate ___ ___________ limiting, not its propagation. Therefore we consider each consecutive stem as a single step towards the final structure. STAR simulates the formation of a RNA structure by adding one stem at a time to the growing structure. Each incorporated stem is the one adding most to the stability of the structure at that point. A true simulation however should take both the rate of formation and the rate of destruction of stems into account. For the sake of simplicity STAR presumes that each intermediate structure is well defined and more or less stable. In this way you can describe the addition of stems to the intermediate structure as a series of linked equilibria. As soon as a local equilibrium is reached, STAR seeks a more favorable one. STAR calculates the release of free energy upon formation of a structure from the Gibbs Free energy values for basepair stacking and loop formation which have been scattered and collected in the literature for the last 20 years. Summarized, the algorithm used to predict a structure is as follows: 1. Find all possible stems of the given RNA sequence. 2. Add stems to the structure until the free energy of the structure can not decrease anymore 2.1 Determine the stem most likely to form next 2.2 If this stem decreases the free energy of the structure: add this stem to the structure 3. Output the structure. - 35 - CHAPTER 3 THE PROCEDURE OF STAR The purpose of STAR is to compute and show a RNA structure. To do that, you should follow a number of consecutive steps. The order of those steps is not arbitrary. Therefore, we list these steps in the following paragraphs. You can see this procedure more detailed in the graph in appendix A. ____ __ ___ ________ _ ________ 3.1 STEP 1: GET YOURSELF A SEQUENCE There are two ways to get a sequence in the computer. The first method is to type in a sequence yourself. You can do this using the option [Primary/Edit]. Look in paragraph 6.6 for an explanation of this option. The other method is to Open a sequence file from disk (use option [Primary/Open]). This sequence file can either be a sequence you have saved once with STAR, or a file from a different origin. For example files from a text editor or a database. You can use all file types here, as long as the contents of the file are coded in ASCII. ____ __ ___ ________ _ _________ 3.2 STEP 2: GET YOURSELF A STRUCTURE When you have a sequence you can get yourself a structure. There are three different methods to do this. The first method is to type one in yourself. You can do this using the option [Structure/Edit]. You find an explanation of this option in paragraph 7.8. The second method is to open a structure file using the option [Structure/Open]. We explain this option in paragraph 7.2. The third method is to let STAR calculate a structure for you. The structure is then based on the previously acquired sequence (step 1). You can do this using the option [Structure/Calculate]. - 36 - Reference Guide STAR Page 3-2 3 The Procedure of STAR 29 June 1990 See paragraph 7.6 for an explanation of this option. - 37 - Reference Guide STAR Page 3-3 3 The Procedure of STAR 29 June 1990 . R E F E R E N C E P A R T - 38 - CHAPTER 4 THE STAR MENU STAR presents the following menubar. +-----------------------------------------------------------+ |Desk Files Primary Secondary Energyrules -.-| +-----------------------------------------------------------+ As the figure above shows, the STAR menubar consists of 6 menus: [Desk],[Files], [Primary], [Secondary], [Energyrules] and [-.-]. The [Desk] menu is not really a STAR menu. It contains the desk accessories that were in the System-file of the diskette you used to start up the computer. In the Atari version of STAR it also contains the [About STAR] option. We explain this option in the chapter that discusses the [Files] menu. From now on we will ignore the [Desk] menu. The [-.-] menu is not a normal menu because its name changes while you use STAR. The purpose of this menu is to show you the status in which STAR is at a certain moment. For example, if you have opened a sequence file, the name of this menu is the name of the file you opened. Unlike the other menus we don't discuss this menu in a separate chapter. We explain it while we discuss the other menus. We explain the other four menus in separate chapters. We discuss these menus briefly in this chapter, so you will understand their organization. - 39 - Reference Guide STAR Page 4-2 4 The STAR menu 29 June 1990 ___ _______ ____ 4.1 THE [FILES] MENU +------------------+----------------------------------------+ |Desk |Files | Primary Secondary Energyrules| +-----------| +-----+----------------------------------+ | Help | |------------| | About STAR | (*1) |------------| | Rename | (*2) | Delete | |------------| | Quit | +------------+ The [Files] menu contains -as suggested by the name- some options that use or handle files. These are [Rename] (*2) and [Delete]. Furthermore, this menu contains some miscellaneous options: [About STAR] (*1), [Quit] and [Help]. See chapter 5 for a detailed explanation of the options in this menu. ------------------------------------------------------------ (*1) For Atari this is an option of the [Desk]-menu. (*2) This is not available on Apple Macintosh. - 40 - Reference Guide STAR Page 4-3 4 The STAR menu 29 June 1990 ___ _________ ____ 4.2 THE [PRIMARY] MENU +---------------------------------+-------------------------+ |Desk Files |Primary | Secondary Energyrules| +-----------------------| +-+-----------------------+ | Help | |-----------| | Open | | Save as...| | Save | |-----------| | View | | Edit | |-----------| | Print | +-----------+ The [Primary] menu contains all the options that operate on sequences, the primary RNA structure. The first option, [Help] gives brief information about the options in this menu. The next three options, [Open], [Save as...] and [Save] let you retrieve or save sequence files. The options [View] and [Edit] are involved with the examination and editing of sequences. The last option, [Print] enables you to make a hardcopy (a print) of a sequence. You find a detailed explanation of the options in this menu in chapter 6. - 41 - Reference Guide STAR Page 4-4 4 The STAR menu 29 June 1990 ___ ___________ ____ 4.3 THE [SECONDARY] MENU +----------------------------------------------+------------+ |Desk Files Primary |Secondary | Energyrules| +-----------------------------------| ++-----------+ | Help | |-----------| | Open | | Save as...| | Save | |-----------| | Options | | Calculate | |-----------| | View | | Edit | |-----------| | Print | +-----------+ The [Secondary] menu contains all the options that operate on the secondary structures of RNA. The first option, [Help] gives brief information about the options in this menu. With the next three options ([Open], [Save as...] and [Save]) you can retrieve or save structure files; you can for example save calculated structures for later use. You will use the following two options ([Options] and [Calculate]) for the calculation (the folding) of the secondary structure. You use the options [View] and [Edit] to examine and to edit (change) a structure. The last option, [Print], enables you make a hard-copy (a print) of the structure. See chapter 7 for a detailed explanation of the options in this menu. - 42 - Reference Guide STAR Page 4-5 4 The STAR menu 29 June 1990 ___ _____________ ____ 4.4 THE [ENERGYRULES] MENU +-----------------------------------------------------------+ |Desk Files Primary Secondary |Energyrules| +-----------------------------------------------| | | Help | |-----------| | Open | | Save as...| | Save | |-----------| | View | | Edit | |-----------| | Print | +-----------+ The options in the [Energyrules] menu involve the energy rules that STAR uses for the calculation of a secondary structure. As you start STAR the program already contains default energy rules. These are the rules as published by Jacobson et al. (*1) with some extensions. See also chapter 3. If you are satisfied with those energy rules, and you don't want to experiment with other energy values, you can ignore the options in this menu. In that case you can skip chapter 9, which explains that menu. The first option in this menu, [Help] gives brief information about the options in this menu. The following three options, [Open], [Save as...] and [Save] allow you to retrieve or save energy rules files. Use the options [View] and [Edit] to examine and to edit (change) energy rules. The final option, [Print], enables you to make a hard-copy (a print) of the energy values. You find detailed explanations of these options in chapter 9. ------------------------------------------------------ (*1) Jacobson,A.B. Good,L. Simonetti,J. Zuker,M. (1984): Nucleic Acids Res., (12)45-52 - 43 - Reference Guide STAR Page 4-6 4 The STAR menu 29 June 1990 ____________ ___ _________ __ ___ __________ ___________ 4.5 SUMMARIZING: THE STRUCTURE OF THE [PRIMARY], [SECONDARY] ___ _____________ _____ AND [ENERGYRULES] MENUS We hope that you have noticed the structure of the most important STAR menus [Primary], [Secondary] and [Energyrules]. The top one is always a [Help] option which gives a brief explanation about the other options in that particular menu. The following three options are always the file-options [Open], [Save as...] and [Save]. Furthermore each menu contains the options [View] and [Edit] to examine and to edit. And the last option in each menu is [Print] which allows you to make a hardcopy. The menu [Secondary] contains two 'extra' options for the calculation of the structure. - 44 - CHAPTER 5 THE [FILES] MENU +------------------+----------------------------------------+ |Desk |Files | Primary Secondary Energyrules| +-----------| +-----+----------------------------------+ | Help | |------------| | About STAR | (*1) |------------| | Rename | (*2) | Delete | |------------| | Quit | +------------+ In this chapter, we fully explain all the options in the [Files] menu. ______ 5.1 [HELP] The [Help] option displays brief information about the options in this menu. The main purpose of this option is to give you some help at your fingertips, without having to consult the manual. Now don't throw away this manual as this option is only a brief reminder. When you press the mousebutton or a key (Apple version) or the [OK] button (Atari version), you leave this option and return to the main menu. ------------------------------------------------------------ (*1) For Atari this is an option of the [Desk]-menu. (*2) This is not available on Apple Macintosh. - 45 - Reference Guide STAR Page 5-2 5 The [Files] menu 29 June 1990 ______ _____ 5.2 [ABOUT STAR] (*1) The [About STAR] option displays some information about the program and the team behind it. Notice that STAR is copyrighted, so please don't make any illegal copies. When you press the mousebutton or a key (Apple version) or the [OK] button (Atari version), you leave this option and return to the main menu. ------------------------------------------------------------ (*1) For Atari this is an option of the [Desk]-menu. - 46 - Reference Guide STAR Page 5-3 5 The [Files] menu 29 June 1990 ________ 5.3 [RENAME] (*1) The [Rename] option enables you to rename a file that is on disk. When you activate this option a file-selector appears, which enables you to select the file that you want to rename. If you are not familiar with the use of a file-selector, consult the manual that was delivered with your computer. After this the file-selector appears again by which you can enter the new filename. From now on, you call the file by its new name. ------------------------------------------------------------ (*1) This option is not available on Apple Macintosh. - 47 - Reference Guide STAR Page 5-4 5 The [Files] menu 29 June 1990 ________ 5.4 [DELETE] The [Delete] option enables you to delete (erase,remove) a file that is on disk. When you activate this option a file-selector appears. With this file-selector you can select the file you want to delete. If you are not familiar with the use of a file-selector, consult the manual that was delivered with your computer. After this the warningbox shown below appears. With this box, STAR asks you to confirm the deletion of the file. +------------------------------+ | Do you really want to delete | | file: 'filename' | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ When you press [OK], STAR deletes the file. If you don't want to delete the file after all, press [Cancel]. - 48 - Reference Guide STAR Page 5-5 5 The [Files] menu 29 June 1990 ______ 5.5 [QUIT] The [Quit] option allows you to stop using STAR the smooth way. This in contrast to the rough way: pressing the reset key or, even worse, turn off the power. When you activate this option the warningbox shown below appears. With this box, STAR asks you to confirm the termination of the program. +-----------------------+ | Do you really want to | | stop this program ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +-----------------------+ If you don't want to stop STAR, press [Cancel]. If you do want to stop STAR, press [OK] and you will return to the desktop. If you made changes to your sequence, structure or energy rules, make sure that you saved them before you press [OK]. Otherwise you will lose them. You can do this with the options discussed in paragraphs 6.3, 6.4, 7.3, 7.4, 9.3 and 9.4. - 49 - CHAPTER 6 THE [PRIMARY] MENU +---------------------------------+-------------------------+ |Desk Files |Primary | Secondary Energyrules| +-----------------------| +-+-----------------------+ | Help | |-----------| | Open | | Save as...| | Save | |-----------| | View | | Edit | |-----------| | Print | +-----------+ In this chapter we fully explain all the options in the [Primary] menu. ______ 6.1 [HELP] The [Help] option displays brief information about the options in this menu. The main purpose of this option is to give you some help at your fingertips, without having to consult the manual. Now don't throw away this manual as this option is only a brief reminder. When you press the mousebutton or a key (Apple version) or the [OK] button (Atari version), you leave this option and return to the main menu. - 50 - Reference Guide STAR Page 6-2 6 The [Primary] menu 29 June 1990 ______ 6.2 [OPEN] The [Open] option enables you to open a sequence file from disk. This means that STAR transfers a copy of a specified sequence from disk to the computer. Then you can use it with STAR. When you activate this option a file-selector appears by which you can select the sequence file you want to open. If you aren't familiar with the use of the file-selector, consult the manual which was delivered with your computer. Note that you can open any file. However not all files contain valid sequences. So be sure that the file you are opening contains a valid sequence. That is why we strongly suggest that you give all your sequence files a recognizable name, for example with the extension '.1'. The extension is the part of a filename after the point '.'. Now you might think why all this trouble in preventing me to open a non-sequence file, why doesn't STAR do that for me? The answer is: in this way you are able to open sequence files originating from databases or from other editors than the STAR editor. This is particularly useful if you want to enter your own sequence, because the STAR editor is very slow. When you have succesfully opened a sequence file, the statusindicator menu shows the name of the opened file. As explained in chapter 4, this is the rightmost STAR menu. If you want to open a sequence file while you already have a sequence and/or a structure in the computer, the following box appears. With this box, STAR asks you to confirm the opening of a (new) sequence file. +------------------------------+ | Opening a sequence will | | overwrite your current | | sequence and structure! | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ If you press [OK], STAR erases the sequence and structure that are in the computer and you are able to open another sequence. This means you lose the sequence and the structure in the _____ computer! So, please be sure that you have saved your current _______ sequence and structure or that they are useless before you press [OK]. You can save your sequence with the options [Save] or [Save as...]. If you are not sure press [Cancel]. Then STAR erases nothing and - 51 - Reference Guide STAR Page 6-3 6 The [Primary] menu 29 June 1990 you return to the main menu. ________ ______ Possible errors WS-FULL A very obvious error occurs when you open a sequence file that is too big for the memory of your computer. This results in a 'WS FULL' error. Unfortunately you can do nothing else than start STAR all over again on a computer with sufficient memory. - 52 - Reference Guide STAR Page 6-4 6 The [Primary] menu 29 June 1990 _____ ______ 6.3 [SAVE AS...] The [Save as...] option enables you to save a sequence to file. This means that STAR transfers a copy of the sequence in the computer's memory to disk. You must enter the name of the file you want the sequence to save to. When you activate this option a file-selector appears which enables you to enter the filename under which STAR will save the sequence. If you are not familiar with the use of the file-selector, consult the manual that was delivered with your computer. Of course there must be a sequence in the computer's memory before you can save it to disk. Therefore you can only activate this option if the computer's memory holds a sequence. This means that either you must have opened (option [Open]) or you must have entered (option [Edit]) a sequence before you can use this [Save as...] option. If you enter a filename that already exists, the box shown below appears. With this box STAR asks you if you want to overwrite the file. +------------------------------+ | File 'filename' | | already exists. Overwrite ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ If you press [OK], STAR saves the sequence to disk. It overwrites the sequence on disk. Otherwise press [Cancel]. Then STAR overwrites nothing and you return to the main menu. When you have saved your sequence, the status-indicator menu shows the filename you entered. - 53 - Reference Guide STAR Page 6-5 6 The [Primary] menu 29 June 1990 ______ 6.4 [SAVE] The [Save] option enables you to save a sequence file for later use. This means that STAR transfers a copy of the sequence in the computer's memory to disk. The difference with the option [Save as...] is that STAR saves the sequence to a file with the same name as the one you opened. That is why you must have opened a sequence file (option [Open]) before you can activate this [Save] option. Furthermore, STAR replaces the sequence on disk by the sequence that you [Save]. So please be careful: be sure that the sequence on disk is no longer useful before you use this option. - 54 - Reference Guide STAR Page 6-6 6 The [Primary] menu 29 June 1990 ______ 6.5 [VIEW] The [View] option enables you to examine the sequence which that is in the computer's memory. This means, of course, that the computer must hold a sequence before you can [View] one. So you must have opened (option [Open]) or created (option [Edit]) a sequence before you can activate this option. Now look as an example at the sequence of the thrT tRNA from ______ E.Coli: 5 10 15 20 25 30 35 40 45 GCUGA UAUAG UUGGU AGAGC CGAGC CUUGG UAAGG GUGAG GUCGG 50 CAGUU CCAAU CUGCC UAUCA GCACC A 100 As you see, STAR divides the sequence into parts of 5 nucleotides. On each line STAR shows 50 nucleotides. The numbers start with 1 at the 5' part of the sequence, and increase until the 3' part of the sequence is reached. - 55 - Reference Guide STAR Page 6-7 6 The [Primary] menu 29 June 1990 ______ 6.6 [EDIT] The [Edit] option enables you to change an existing sequence, or create a new sequence. We fully explain the editor in the following paragraphs. Unfortunately this editor is quite slow. This is inevetable with the facilities of the language in which we wrote STAR (APL). If you don't want to use this option because of its speed, you can edit sequences with another text editor. You can use a file originating from such an editor using the option [Open] (see paragraph 6.2). ________ ___ ______ 6.6.1 Entering The Editor You enter the STAR editor by activating the option [Edit] from the [Primary] menu. If your sequence is less than 901 nucleotides (Atari ST) or less then 1051 nucleotides (Apple Macintosh) long, you enter the editor without further ado. However, if your sequence is longer, then the box shown below appears. It enables you to edit a big sequence by working on a smaller part. +---------------------------------------+ | Sequence too big to fit in editor | | | | Edit which part ? | | | | Edit nucleotide ___ to ___ | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------- | +---------------------------------------+ You must enter the beginning and end positions of the nucleotides of that part of the sequence you want to edit at the fields marked by __. For example, if you want to edit the first 100 nucleotides of a sequence 2000 nucleotides long, type 1 at the first field, and 100 at the second one. After you have entered these values, press [OK]. If you don't want to edit the sequence, press [Cancel]. ________ ______ Possible errors If you enter an illegal range in the box above, or you enter other characters than numbers, the box appears again. In fact the box keeps appearing until you enter a correct range. - 56 - Reference Guide STAR Page 6-8 6 The [Primary] menu 29 June 1990 ___ ______ ______ ___ ______ 6.6.2 The Editor Screen And Cursor The editor shows you the sequence in the same way as the [View] option (paragraph 6.5) does. The cursor of the editor is the blinking dash. The cursor indicates whether you are working in insert or in overwrite mode. If the cursor is a high dash, you are in overwrite mode. If it is a low dash, you are in insert mode. We explain the meaning of the insert and overwrite mode in paragraph 6.6.3.3 ___ ______ ____ 6.6.3 The Editor Keys You can use several keys during editing. We list them in the following paragraphs. ______ _______ ___ ________ 6.6.3.1 Moving Through The Sequence To move through the sequence, you can use the arrowkeys. As their use is obvious, we don't discuss them further. If you want to move faster through the sequence, you can use the [Tab] key. If you press this key, the cursor moves to the beginning of the next group of five nucleotides. Another way to move through the sequence is by using the mouse. ________ _ __________ 6.6.3.2 Deleting A Nucleotide You can use two different keys to delete a nucleotide; the [Delete] key or the [Backspace] key. If you press [Delete] STAR deletes (erases) the nucleotide at the cursor position . If you press [Backspace], STAR deletes the nucleotide just left of the cursor. ______ ___________ 6.6.3.3 Adding Nucleotides You can add nucleotides by typing in the letter corresponding with that particular nucleotide. - 57 - Reference Guide STAR Page 6-9 6 The [Primary] menu 29 June 1990 The keys that correspond with each type of base are: A:Adenine C:Cytidine U:Uracil G:Guanine You can enter the nucleotides both in lowercase and uppercase. Remember however that STAR calculate a structure of uppercase typed nucleotides only. This feature is particularly useful if you want to exclude a certain part of a sequence to basepair. You exclude that part by typing it in lowercase. Normally you are advised to type with the [CapsLock] key pressed. See for more information paragraph 7.6. When you are in 'insert' mode, STAR inserts the entered nucleotide just left of the cursor position. In 'overwrite' mode, the entered nucleotide replaces the nucleotide at cursor position. See also paragraph 6.6.2 and paragraph 6.6.3.4. When your sequence grows longer then 900 nucleotides for Atari users or 1050 nucleotides for Apple users, STAR is unable to handle your complete sequence. You should terminate your session and resume editing with a smaller part of the sequence. STAR warns you with the following box, and you should leave the editor and resume editing with a smaller part of the data. +----------------------------------+ | The object you are editing | | is grown too big: it doesn't | | fit in the editor anymore | | | | You must either [Exit] or [Quit] | | from the editor. | | | | -------+ +------+ | | | Exit | | Quit | | | +------+ +------- | +----------------------------------+ Pressing the [Exit] button has the same effect here as activating the [File/Exit] option from the menubar. This means that you leave the editor and all changes that you made will be fixed. Pressing the [Exit] button has the same effect as activating the [File/Exit] option from the editormenubar; you leave the editor and all the changes you made are fixed. Pressing [Quit] has the same effect as activating the [File/Quit] option from the editor menubar. In this case you will also leave the editor, but you lose all the changes that you made during the editing session. Once you have left the editor, you can resume editing, though part-wise only; see paragraph 6.6.1. - 58 - Reference Guide STAR Page 6-10 6 The [Primary] menu 29 June 1990 ______ _______ ________ ___ ___________ ____ 6.6.3.4 Toggle Between 'insert' And 'overwrite' Mode You can toggle between 'insert' and 'overwrite' mode using the [Insert] key. You see the cursor change from high dash to low dash or vice versa. When the cursor is a high dash you are in overwrite mode, when it is a low dash you are in insert mode. See also paragraph 6.6.2 and paragraph 6.6.3.3. - 59 - Reference Guide STAR Page 6-11 6 The [Primary] menu 29 June 1990 ___ ______ ____ 6.6.4 The Editor Menu +----------------------------------+ |Desk File Clipboard | +----------------------------------+ When you enter the editor the menubar as shown above appears. As you see, the menubar consists of three menu's: [Desk],[File] and [Clipboard]. The [Desk] menu is not really a STAR menu. It contains the desk-accessories that were in the System-file of the diskette you used to start the computer. In the Atari version it also contains the [About STAR] option. We discussed this option in paragraph 5.2. From now on we ignore the [Desk] menu. However, we deal with the other two menus in the following paragraphs. ______ 6.6.4.1 [File] +------------------+---------------+ |Desk |File | Clipboard | +-----------| +-+-------------+ | Replace| | Glue | |--------| | Exit | | Quit | +--------+ The first two options in the [File] menu, [Replace] and [Glue] are miscellaneous editor-options. You can use the last two options to leave the editor and return to the main menu. We fully discuss these options in the following paragraphs. _________ 6.6.4.1.1 [Replace] _______ You can use the [Replace] option to replace one type of ______ nucleotide into another type. You can also use it to delete all nucleotides of a certain type in the sequence. Let us explain this by an example. Suppose you have a sequence ___ file which you obtained from an (ASCII) DNA databank. You can open this file using the option [Primary/Open] (see paragraph 5.2). Now you want to use STAR to fold this sequence. However ___ STAR is only able to fold RNA sequences. Therefore you must replace all T bases in the DNA sequence by U's. You can use the [Replace] option to do this. When you activate this option the box shown below appears. With - 60 - Reference Guide STAR Page 6-12 6 The [Primary] menu 29 June 1990 this box you can enter the nucleotides to replace, and the nucleotides you want to replace them with. +--------------------+ | Replace T by U | | | | | (type # to delete) | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------+ Enter the nucleotides on the fields marked by __. You can move the cursor between both fields using the mouse, or the [Tab] and [Ctrl/Tab] keys. We placed the nucleotides T and U as default in the box. If you press [OK], STAR replaces the nucleotides. If you have changed your mind, press [Cancel]. Then STAR replaces nothing and you return to the main editor menu. If you type a # (a hash) as the replacement nucleotide (the rightmost field), STAR deletes all nucleotides of the type given in the leftmost field. For instance, if you want to delete all A's in your sequence, you enter: +--------------------+ | Replace A by # | | | | | (type # to delete) | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------+ ________ ______ Possible errors What happens if you enter a nucleotide to be replaced that does not exist in your sequence? For instance you type: +--------------------+ | Replace Z by # | | | | | (type # to delete) | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------+ If there is no Z in the sequence, the following box appears: - 61 - Reference Guide STAR Page 6-13 6 The [Primary] menu 29 June 1990 +--------------+ | Character to | | be replaced | | not found | | | | +------+ | | | OK | | | +------+ | +--------------+ You simply press [OK] (STAR erased nothing) and you leave the [Replace] option and return to the main editor-menu. ______ 6.6.4.1.2 [Glue] You can only use this option while you are editing a structure, see paragraph 7.8.4.1.2 ______ 6.6.4.1.3 [Exit] Use the [Exit] option to leave the editor and return to the main menu if you are satisfied with your changes. All changes that you made during the edit session will be fixed. If you don't want this, you can use the option that we discuss in the next paragraph: [Quit] If you created a new sequence during editing, the statusindicator menu shows '-.E' when you have left the editor. In this way STAR shows you that you have a sequence in the memory of your computer, although you did not [Open] a sequence file, but entered it with the editor. ______ 6.6.4.1.4 [Quit] Like in the previous paragraph, you can use the [Quit] option to leave the editor and return to the main menu. However, when you ___ use this option, STAR will not fix the changes you made during the editing of the sequence. This means that you lose all changes that you made during the edit session! That is why after activation of this option the warning-box shown below appears. With this box STAR asks you to confirm the quitting from the editor. - 62 - Reference Guide STAR Page 6-14 6 The [Primary] menu 29 June 1990 +--------------------------+ | You are about to QUIT | | from the editor | | ALL CHANGES WILL BE LOST!| | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------------+ If you press [OK], you leave the editor without saving the changes. If you press [Cancel] you don't leave the editor and return to the main editor menu. - 63 - Reference Guide STAR Page 6-15 6 The [Primary] menu 29 June 1990 ___ ___________ ____ 6.6.4.2 The [Clipboard] Menu +----------------------------------+ |Desk File |Clipboard | +-----------------------| ++ | Cut | | Copy | | Paste | |---------| | Unblock | +---------+ The [Clipboard] menu contains all options that use the clipboard. You are probably familiar with the use of it. If you are not, don't despair as we discuss the basics of using the clipboard below. The clipboard is a piece of memory in your computer into which you can move or copy large pieces of your sequence. The way it works is that you first put a part of the sequence in the clipboard using either the [Cut] or the [Copy] option. Later on you can insert that part anywhere in your sequence using the [Paste] option. In the following paragraphs we discuss these options. _____ 6.6.4.2.1 [Cut] You can use the [Cut] option to transfer nucleotides from the STAR editor to the clipboard. First you select a part of the sequence by dragging the mouse over the sequence. Dragging means that you point the mouse at the first nucleotide, push the mousebutton and move the mouse to the last nucleotide. During this you must keep the mousebutton pressed. When you now release the mousebutton, STAR will select the part of the sequence. You can see this because it is now shown in 'reversed video'. This means white letters on a black background. The [Cut] option puts the selected part of the sequence in the clipboard, and remove the selected part from the editor. If you don't want to erase the part of the sequence, you use the following option: [Copy]. ______ 6.6.4.2.2 [Copy] Similar to the [Cut] option, you can use the [Copy] option to transfer nucleotides from the STAR editor window to the clip- - 64 - Reference Guide STAR Page 6-16 6 The [Primary] menu 29 June 1990 board. First you select a part of the sequence by dragging the mouse over the sequence. If you are not familiar with the term 'dragging' see the previous paragraph, as we explain it there. The [Copy] option places a copy of the selected part of the sequence into the clipboard. STAR doesn't remove the selected part. If you do want to remove the selected part, you should use the [Cut] option. This option is discussed in the previous paragraph. _______ 6.6.4.2.3 [Paste] You can use the [Paste] option to transfer nucleotides from the clipboard back to the STAR editor. STAR doesn't erase the string in the clipboard, so you can [Paste] the same string several times. When your sequence grows longer then 900 nucleotides for Atari users or 1050 nucleotides for Apple users, STAR is unable to handle your sequence in total anymore. So you must terminate your session and resume editing with a smaller part of the sequence. STAR warns you with the box that we explain in paragraph 6.6.3.3. _________ 6.6.4.2.4 [Unblock] You can use the [Unblock] option to erase the current block-definition. This is indicated by the stems which STAR shows in normal video again (black letters on a white background). Other ways to erase the blockdefinition are to press the mousebutton once, or to press any key. - 65 - Reference Guide STAR Page 6-17 6 The [Primary] menu 29 June 1990 _______ 6.7 [PRINT] Use the [Print] option to make a hard-copy (a print) of your sequence. You can only activate this option if you have a sequence in your computer's memory, of course. When you activate this option one of the following boxes appears, depending on whether your computer is an Atari or an Apple. +------------------------------+ ----------------------------+ | PRINT OPTIONS | | PRINT OPTIONS | | | | | | Page length __ | | Printer o Image o Deskjet | | Page width __ | | | | Top margin __ | | Page length __ | | Bottom margin __ | | Page width __ | | | | Top margin __ | | +-------+ --------+ | | Bottom margin __ | | Print to:|Printer| | File | | | | | +-------- +-------- | | Print to: o Printer o File| | | | | | -------+ +------+ | | -------+ +------+ | | |Cancel| | OK | | | |Cancel| | OK | | | +------+ +------- | | +------+ +------- | +------------------------------- +---------------------------+ Atari box Apple box In the four fields marked by __, you may enter the length and width of the paper you use, the top margin and the bottom margin. The top margin is the blank part above the text on a page. The bottom margin is the blank part below the text on a page. You enter all these settings in number of characters. For instance, if you use paper 71 characters long, enter '71' at the first field. For a standard A4 paper format, you enter '66' for the pagelength, and '80' for the page width. For standard continuous forms paper, enter '71' for the pagelength and '80' for the pagewidth. We advise for both top and bottom margin a length of 2 characters. If you use a MacIntosh you should specify the type of your printer. If it is an Apple ImageWriter, you select the [Image] button. If it is a HP Deskjet+, you select the [Deskjet] button. HP Deskjet+ users should also set DIP-switch number 5 from the rightmost DIP-switch block to "on". This DIP-switch block is positioned at the front of the printer. Both Atari and Apple users can specify whether they want to print to a printer or to a file. Printing to a printer means that STAR outputs directly to a printer. In this case a printer must be attached to your computer and it must be switched on and be on-line. If you don't have a printer attached, but a printer attached to - 66 - Reference Guide STAR Page 6-18 6 The [Primary] menu 29 June 1990 another computer is available, you can print to file. In this case STAR asks you to enter a filename using a file-selector. If you are not familiar with the use of a file-selector, please consult the manual that was delivered with your computer. Now STAR will make a printerfile for you. ________ ______ Possible errors: If you specify you want to print to a printer while there is no printer attached to your computer, you can not use STAR for about |0 seconds. - 67 - CHAPTER 7 THE [SECONDARY] MENU +----------------------------------------------+------------+ |Desk Files Primary |Secondary | Energyrules| +-----------------------------------| ++-----------+ | Help | |-----------| | Open | | Save as...| | Save | |-----------| | Options | | Calculate | |-----------| | View | | Edit | |-----------| | Print | +-----------+ In this chapter we fully explain all the options in the [Secondary] menu. - 68 - Reference Guide STAR Page 7-2 7 The [Secondary] menu 29 June 1990 ______ 7.1 [HELP] The [Help] option displays brief information about the options in this menu. The main purpose of this option is to give you some help at your fingertips, without having to consult the manual. Now don't throw away this manual as this option is only a brief reminder. When you press the mousebutton or a key (Apple version) or the [OK] button (Atari version), you leave this option and return to the main menu. - 69 - Reference Guide STAR Page 7-3 7 The [Secondary] menu 29 June 1990 ______ 7.2 [OPEN] The [Open] option enables you to open a structure file from disk. This means that STAR transfers a copy of a specified structure from disk to the computer. Then you can use it with STAR. When you activate this option a file-selector appears by which you can select the structure file you want to open. If you don't know how to use the file-selector, consult the manual that was delivered with your computer. A structure is closely related to the sequence from which it originates. Therefore you can only open structure files whose filename is the same as the filename of the sequence you have opened. As a structure is very specific for STAR, you can only open structure files that you once saved by STAR. To prevent opening other files, you can only open files with an extension beginning with '2'. The extension is the part of the filename after the point '.'. For example: you opened the sequence file 'TMV.1'. Now you can open only structure files beginning with 'TMV.2'. The remainder of the filename is not limited. For example: you can open the structure file 'TMV.2A'. If you try to open a file with a wrong filename, the box shown below appears. With this box STAR warns you that you can not open the file. +------------------------------+ | Can not open file: | | 'wrong filename' | | Only 'right filename'allowed | | | | +------+ | | | OK | | | +------+ | +------------------------------+ If you try to open a file that is not a structure file (only possible on Atari), the box shown below appears. With this box STAR warns you that the file you try to open is not a structure file. +--------------------+ | This file is not a | | structure file! | | | | +------+ | | | OK | | | +------+ | +--------------------+ All you can do is press [OK], and you return to the main menu. - 70 - Reference Guide STAR Page 7-4 7 The [Secondary] menu 29 June 1990 If you want to open a structure file, while you already have a structure in the computer, the box shown appears. With this box STAR asks you to confirm the opening of a (new) structure file. +------------------------------+ | Opening a structure file | | will overwrite your current | | structure! | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ If you press [OK], STAR erases the structure that is in the computer and you are able to open another structure. This means you lose the structure in the computer! So, please be sure that _____ _______ you have saved your current structure or that it is useless before you press [OK]. You can save your structure with the options [Save] or [Save as...]. If you are not sure press [Cancel]. Then STAR erases nothing and you return to the main menu. ________ ______ Possible errors WS-FULL A very obvious error is when you want to open a structure file that is too big for the memory of your computer. This results in a 'WS FULL' error. Unfortunately you can do nothing else than start STAR all over again on a computer with sufficient memory. - 71 - Reference Guide STAR Page 7-5 7 The [Secondary] menu 29 June 1990 _____ ______ 7.3 [SAVE AS...] The [Save as...] option enables you to save a structure file. This means that STAR transfers a copy of the computed structure to disk. You enter the name of the file you want the structure to save to. When you activate this option a file-selector appears by which you can enter the filename under which STAR will save the structure. If you are not familiar with the use of the file-selector, please consult the manual which was delivered with your computer. Of course the computers must have a structure in memory before you can save it to disk. Therefore you can only activate this option if your computers memory holds a structure. This means that you must either have opened (option [Open]) or entered (option [Edit]) a structure before you can use this option. As a structure is closely related to the sequence from which it originates, you can only save to a file with a special name. The first part of the filename must be equal to the first part of the name of the sequence file you opened. With the first part of a filename we mean the part of the name preceding the point '.'. Furthermore, the extension of the filename must begin with '2'. The extension of a filename is the part of the name following the point '.'. For example: you opened the sequence 'TMV.1'. Now you can save your structure only to a file beginning with 'TMV.2'. The remainder of the filename is not limited. For example: you can save the structure to a file named 'TMV.2A'. If you enter a filename that is not allowed, the box shown below appears. With this box STAR warns you that the filename is not allowed and that it did not save the structure. +------------------------------+ | Filename:'wrong filename' | | is not allowed. | | Structure not saved. | | | | +------+ | | | OK | | | +------+ | +------------------------------+ All you can do is press [OK] and you return to the main menu. Then you can use the option [Save as...] again to save to a file with another filename. If you enter a filename that already exists, the box shown below appears. With this box STAR asks you if you want to overwrite the file. - 72 - Reference Guide STAR Page 7-6 7 The [Secondary] menu 29 June 1990 +------------------------------+ | File 'filename' | | already exists. Overwrite ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ If you press [OK], STAR saves the structure to disk. It overwrites the structure on disk. Otherwise press [Cancel]. Then STAR overwrites nothing and you return to the main menu. When you have succesfully saved your structure, the rightmost part of the status-indicator menu shows '/2xx'. 'xx' is the part of the extension of the structure file that is not limited. For example, you opened the sequence file 'TMV.1' and you saved a structure to a file named 'TMV.2A'. Then the status-indicator menu shows 'TMV.1/2A'. - 73 - Reference Guide STAR Page 7-7 7 The [Secondary] menu 29 June 1990 ______ 7.4 [SAVE] The [Save] option enables you to save a structure file for later use. This means that STAR transfers a copy of the computed structure to disk. The difference with the option [Save as...] is that STAR saves the structure to a file with the same name as you opened. That is why you should have opened a structure file (option [Open]) before you can activate this option. Furthermore, STAR replaces the structure on disk by the recently computed structure without warning. So be careful with this option: be sure that the structure on disk is no longer useful before you use this option. - 74 - Reference Guide STAR Page 7-8 7 The [Secondary] menu 29 June 1990 _________ 7.5 [FACTORS] The [Factors] option enables you to change the default values for some parameters that STAR uses for the calculation of a structure. These parameters are: Sequential folding factor, Temperature folding factor and Number of stems. You can change these parameters using the box shown below. This box appears when you activate the [Factors] option. +------------------------------------------------+ | Folding parameters | | | | Sequential folding factor 0__________ | | | | Temperature folding factor 0__________ | | | | Number of stems 1000_______ | | | | | | +------+ +------+ | | | OK | |Cancel| | | +------+ +------- | +------------------------------------------------+ You can enter the parameters on the fields marked by __. As you can see we have already set parameters as default: 0 for sequential and temperature folding factor and 1000 for number of stems. To change the parameters you change the default parameters and press [OK]. If you don't want to change them, press [Cancel] We discuss the parameters in the following paragraphs. __________ __________ _______ ______ 7.5.1 [Factors]: Sequential Folding Factor If a RNA molecule is synthesized the 5' terminus is the first one formed. You can imagine that stems that are formed near to the 5' terminus are favored above stems that are formed near to the 3' terminus. So the RNA starts the folding at the 5' terminus of the molecule. This process is called 'sequential folding' in the literature. STAR can deal with sequential folding during the simulation of the folding of the RNA molecule. Stems near to the 5' terminus can get a bonus so that STAR adds them earlier to the structure. You can define this bonus by entering a value bigger then 0 for the Sequential folding factor in the box discussed in the previous paragraph. The bigger you define the sequential folding factor, the bigger the bonus. - 75 - Reference Guide STAR Page 7-9 7 The [Secondary] menu 29 June 1990 STAR calculates this bonus in the following way: B = (SFF / 100) x N where... B = bonus SFF=sequential folding factor N = nucleotide number of the most 5' nucleotide of the stems (the fourth column in the definition of a stem; see paragraph 7.7.1). STAR adds this bonus to the free energy of the basepair of the stems (the fifth column in the definition of a stem; see paragraph 7.7.1). __________ ___________ _______ ______ 7.5.2 [Factors]: Temperature Folding Factor During and after the time a RNA molecule is synthesized, the RNA folds into its native structure. You can imagine that the chance that two nucleotides in a sequence meet each other to form a basepair depends on two factors: 1. the distance (in number of nucleotides) between the two nucleotides. This is the length of the loop formed. 2. a hypothetical factor that describes the flexibility of the RNA molecule. As we see it, this factor depends on the temperature of the RNA. The first factor, distance, is dealt with in the energy gain/loss calculations. The energy needed to form a hairpin, bulge or interior loop is higher if the loop length is greater. The second factor, 'the temperature folding factor', is theoretical. Default we have set this factor to 0 so as to have no effect. You can change this factor using the box discussed in paragraph 7.5. When you set this factor to a value higher than 0, long distance interactions are less favorable. When you set the factor to a value lower than 0 these interactions are more favorable. STAR uses this factor during the prediction of a structure by multiplying the energy of the single stranded stretch with EXP(TFF / 1000) where... EXP=exponential TFF=Temperature Folding Factor - 76 - Reference Guide STAR Page 7-10 7 The [Secondary] menu 29 June 1990 __________ ______ __ _____ 7.5.3 [Factors]: Number Of Stems As we discussed in paragraph 2.5, the first step of STAR folding a sequence is the generation of all possible stems by the given sequence. However the memory of a computer is limited. So if STAR would generate all possible stems the suitable memory would be exceeded. Especially when STAR is folding a large sequence. Therefore you can limit the maximum number of generated stems by the box discussed in paragraph 7.5. The stems that STAR then generates are the ones with the lowest free energy. However, you can not be sure that a stem that is ___ not generated now because of the limited number of stems, should not decrease the free energy of the total structure. Furthermore many of the generated stems exclude each other. Therefore you should always set this parameter as highly as possible regarding the memory of your computer. Let us try make this clearer with an example. Suppose you let STAR calculate (fold) a hypothetical sequence. You set the 'Maximum number of stems' parameter to 5. Suppose now that STAR finds the following five stems: 1 5 10 15 | 2 6 11 16 | increasing free energy 3 7 12 17 | 4 8 13 18 | (dG less negative) 20 25 30 35 V If you don't know the meaning of these values, check paragraph 7.7.1. (the energy values of the hypothetical stems are not included). Because some stems overlap, STAR incorporates the first and the last stem into the final structure. This structure is now: 1 5 10 15 20 25 30 35 Now you let STAR fold the same sequence, but with the 'Number of stems' parameter set to 4. In this case STAR will find the following four stems: 1 5 10 15 | increasing free energy 2 6 11 16 | 3 7 12 17 | 4 8 13 18 V (dG less negative) Again, most stems overlap, but now STAR only incorporates one stem in the final structure. This structure is now: 1 5 10 15 So the final structure is different from the one in the previous example: one stem is 'missing'. Furthermore, because one stem decreasing the amount of free energy of the structure is not included, the total structure is less stable. This means that - 77 - Reference Guide STAR Page 7-11 7 The [Secondary] menu 29 June 1990 the chance that the calculated structure is the same as the one found in nature is smaller. In other words: the reliability is smaller. The default for the 'Number of stems' parameter is 1000. After calculating many structures, we had good results using this default. However, as said before, if the memory of your computer is sufficient you better set this parameter to a higher value. This increases the reliability of the calculation. - 78 - Reference Guide STAR Page 7-12 7 The [Secondary] menu 29 June 1990 ___________ 7.6 [CALCULATE] [Calculate] is the essential option of STAR. It calculates (predicts, folds) a structure for you. You should have a sequence in your computer's memory before you can use this option. In the now following paragraphs we follow the calculation step by step. In the first paragraph we tell you what to do before you start the calculation. In the second paragraph we tell you how to start the calculation. In the third and fourth paragraphs we follow the folding process. Finally in the fifth paragraph we tell you how STAR shows the calculated structrure. ______ ___ _____ ___ ___________ 7.6.1 Before You Start The Calculation Before you start the calculation be sure that you have a sequence in the computer. If you don't, get one using the options [Primary/Open] or [Primary/Edit]. Also, be assured that you are satisfied with the folding parameters. See the previous paragraph (paragraph 7.5). Furthermore, are you satisfied with the default energy rules? If you are not, you can change them by opening an existing energy rules file (option [Energyrules/Edit] or edit the default energy rules (option [Energyrules/Edit]). When you are sure that certain stems must exist in the structure, you can force STAR to incorporate those stems in the structure. If you want that, you simply define those stems before you start the calculation. We explain this procedure in paragraph 7.7 where we discuss the option [Edit]. Remember that STAR will also incorporate the stems of an already existing structure in the new structure. So if you already have a structure in your computer's memory and you don't want the stems of that structure to be incorparated in the new structure, you must erase the stems of the existing structure before you start the calculation. You can do this with the [Edit] option (paragraph 7.7). When you are sure that a certain part of the sequence does not basepair with the rest of the sequence, you can prevent the basepairing of this region. If you want that, you should change the nucleotides of that part of the sequence to anything other than A,G,C or U. You can do this using the option [Primary/Edit]. We advise you to replace the uppercase letters by lowercase. Then you will be able to change the nucleotides back to normal using the [Primary/Edit/Replace] option. - 79 - Reference Guide STAR Page 7-13 7 The [Secondary] menu 29 June 1990 ________ ___ ___________ 7.6.2 Starting The Calculation You start the calculation by activating the option [Calculate]. When there is already a structure in the memory of the computer, the box shown below appears. With this box STAR warns you that it will overwrite your current structure if you let it calculate a new structure. +------------------------------+ | Predicting a structure now | | will add stems to your | | current structure | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ When you are sure that stems can be added to the structure you already have, you can press [OK]. Otherwise press [Cancel]. Then you will return to the main menu so you can save that structure for later use. You can do this by the options [Save as] or [Save]. Remember that STAR will incorporate the stems of the already existing structure in the new structure (see previous paragraph). If you don't want that, you should erase the stems of the existing structure before you start the calculation. You can do this with the [Edit] option (paragraph 7.7). Now the calculation starts. __________ __ ___ ________ _____ 7.6.3 Generation Of All Possible Stems As mentioned in paragraph 2.5, STAR first generates all possible stems of your sequence. Remember that 'all possible stems' also depends on the 'Max number of stems' parameter; see paragraph 7.5.3. STAR generates those stems by storing all possible direct stackings of a sequence in a stacking table. Because the memory of a computer is limited, STAR doesn't create this table in one run. During this process, STAR displays the size of the subtable that is formed at a certain moment. For example: Size of stack-table being searched: 90 59 (run 1) Size of stack-table being searched 32 32 (run 2) When STAR has found all possible stems, STAR tells you how many stems it has found, for example: 229 stems found, they are now being processed. - 80 - Reference Guide STAR Page 7-14 7 The [Secondary] menu 29 June 1990 ___ __________ __ ___ _________ _____ 7.6.4 The Processing Of The Generated Stems After the generation of all possible stems STAR processes them. This means that STAR adds the most stable stems one by one to the structure. During this process, STAR displays each stem it adds at a certain moment, for example: 27 32 38 43 15.9 4.1 added to structure (first stem added) 49 53 61 65 12.0 4.7 added to structure (second stem added). In the next paragraph we explain the meaning of this values. ___ __________ _________ 7.6.5 The Calculated Structure When STAR has processed all stems, the calculation process is finished. Now STAR shows the generated structure in the following way: 27 32 38 43 * -15.9 4.1 * CACCCU * GUGGGA 49 53 61 65 * -12.0 4.7 * GGCAG * CCGUC 1 7 66 72 * -14.4 4.6 * CGUGAUA * CGACUAU 11 13 22 24 * -4.2 5.0 * CUC * GAG We explain the meaning of the values in this table in paragraph 7.7.1 - 81 - Reference Guide STAR Page 7-15 7 The [Secondary] menu 29 June 1990 ______ 7.7 [VIEW] The [View] option enables you to examine the structure that is in memory. Therefore, you can only activate this option if there is a structure present in the computer. So you must either have opened (option [Open]), entered (option [Edit]) or calculated (option [Calculate]) a structure before you can activate this option. After activating this option one of the boxes shown below appears, depending on whether your computer is an Atari or an Apple. With this box you can select the way you want to view the structure. +-------------------------+ --------------------------+ | View structure as | | View structure as | | | | | | -------+ ------+ -----+ | | | | |Table | |Span | |Lego| | | o Table o Span | | +------- +------ +----- | | | | | | | | ---------+ | | | | |Mountain| | | o Lego o Mountain | | +--------- | | | | | | | | -------+ +--------+ | | -------+ +--------+ | | | OK | | Cancel | | | | OK | | Cancel | | | +------+ +--------- | | +------+ +--------- | +-------------------------- +-------------------------+ Atari box Apple box There are 4 ways to view a structure: 1) by table, 2) by span-view, 3) by lego-view and 4) by mountain-view. ___ ______ ___ _________ ___ ____ __ ____ ____________ N.B. The table- and span-view are able to show pseudo-knots __________ ___ _____ ___ _____________ ____ ____ __ _________ correctly, the lego- and mountain-view will show an incorrect _______ __ ____ __ _____________ picture in case of pseudo-knots. You can use the socalled "radio-buttons" [Table], [Span], [Lego] and [Mount] to select which way you want to view the structure. When you press [OK] STAR displays the structure. If you press [Cancel], you return to the main menu. The following paragraphs illustrate these outputs with the ______ example of thrT tRNA from E.Coli. ____ _________ __ _____ 7.7.1 View Structure As Table When you view a structure as [Table], the following table appears on screen: - 82 - Reference Guide STAR Page 7-16 7 The [Secondary] menu 29 June 1990 27 32 38 43 * -15.9 4.1 * CACCCU * GUGGGA 49 53 61 65 * -12.0 4.7 * GGCAG * CCGUC 1 7 66 72 * -14.4 4.6 * CGUGAUA * CGACUAU 11 13 22 24 * -4.2 5.0 * CUC * GAG In this table, the first four columns define the location of a stem: - Column 1 contains the base number of the most 5' nucleotide of the stem. - Column 2 contains the base number of the 3' end of the 5' stem-half. - Column 3 contains the base number of the 5' end of the 3' stem-half. - Column 4 contains the base number of the most 3' nucleotide of the stem. The next two columns show the energy content of the stem: - Column 5 contains the Gibbs free energy of the base pairs in kcal/mol. - Column 6 contains the Gibbs free energy of the single stranded stretch of RNA located directly between the corresponding bases of the second and third column in kcal/mol. At the rightmost side of the table you see the basepairings of the corresponding stem. ____ _________ __ _________ 7.7.2 View Structure By Spannings When you view a structure by [Span], the following picture appears on screen (after a while): ------------------------------------------------------------ | ---------------- ----------------- ------------ | | | | | | |-----| |--| |--| |----| |----| |----| GCUGAUAUAGCUCAGUUGGUAGAGCGCACCCUUGGUAAGGGUGAGGUCGGCAGUUCCAAU : : : : : : : : : : : : 123456789012345678901234567890123456789012345678901234567890 1 2 3 4 5 6 - 83 - Reference Guide STAR Page 7-17 7 The [Secondary] menu 29 June 1990 ------------- <-- ----- | <-- stems | | <-- |---||------| <-- CUGCCUAUCAGCACCA <-- sequence : : : 1234567890123456 <-- nucleotide- 7 <-- numbers In the middle row of this picture, you see the nucleotides. Just below that, you see a ':' every fifth nucleotide. And the two bottom lines are the nucleotides numbers: in the bottom line the units and one line above the tens. The nucleotides are numbered starting at 1 at the 5' end and increase until the 3' end is reached. Above the sequence you see the stems drawn. STAR draws a stem by connecting two singlestranded stemhalfs. STAR draws a stemhalf as: |----| STAR draws a stem as: ------------------ | | |----| |----| This presentation is more visual than the alternative method: 'view as Table'. The disadvantage of this 'graphic view' is however that STAR takes more time to calculate it. Furthermore it gives less information than the table as the energy values are not included. ____ _________ __ ________ 7.7.3 View Structure As Mountain When you show a structure by mountain-view, you see the following picture. - 84 - Reference Guide STAR Page 7-18 7 The [Secondary] menu 29 June 1990 UGGUA U--|--A UUCCAAU AGUUGGUA C---|---G G---|--- C----|---G C----|----G A----|--- U-----|----A C-----|-----G C-----|--- C------|-----G A------|------U G------|--- UAG-------|------CGC-------|-------GAGGUCG-------|--- A-----------------------------|----------------------- U------------------------------|----------------------- A-------------------------------|----------------------- G--------------------------------|----------------------- U---------------------------------|----------------------- C----------------------------------|----------------------- G-----------------------------------|----------------------- : : : : : : : : : : : : 123456789012345678901234567890123456789012345678901234567890 1 2 3 4 5 6 C -U --G ---C ----C -----U ------A -------U --------C ---------A ----------G -----------CACCA : : : 1234567890123456 7 Basically this is the representation as developed by Hogeweg & Hesper (*1). Pairing nucleotids are connected by horizontal lines; the centre is indicated by a "|". N.B. Remember that pseudoknots will dislay an incorrect picture if you use the mountain-view. ____ _________ __ ____ 7.7.4 View Structure As Lego When STAR shows a structure as Lego-view, you see the following picture. ------------------------------------------------------------ (*1) Hogeweg,P. Hesper,B.(1984): Energy directed folding of RNA sequences. Nucleic Acids Research 12, p.67-74. - 85 - Reference Guide STAR Page 7-19 7 The [Secondary] menu 29 June 1990 UGGUA U-|-A UUCCAAU AGUUGGUA C-|-G G--|--C C---|--G C-|-G A--|--U U---|--A C-|-G C--|--G C---|--G A-|-U G--|--C UAG---|--CGC-|-GAGGUCG--|--C A-------------|------------U U-------------|------------A A-------------|------------U G-------------|------------C U-------------|------------A C-------------|------------G G-------------|------------CACCA This lego-view was derived by F.H.D.van Batenburg from the mountain-view in order to reduce the mountainous size of the that view. Nevertheless it presents the same information as the classical mountain-view. Remember that pseudoknots will display an incorrect picture if you use the lego-view. - 86 - Reference Guide STAR Page 7-20 7 The [Secondary] menu 29 June 1990 ______ 7.8 [EDIT] The [Edit] option enables you to change an existing structure or create a new structure. This is particularly useful if you want to force STAR to incorporate certain stems into the predicted structure (see paragraph 7.6.1). You can activate this option only if there is a sequence present in the computer. A structure may or may not be present. If it is, you will be able to change the present structure. If it is not, you can enter a new structure. We fully explain the editor in the following paragraphs. Unfortunately this editor is quite slow. This is inevitable with the language facilities in which we wrote STAR (APL). So please be patient while you are editing. ________ ___ ______ 7.8.1 Entering The Editor You enter the editor by activating the option [Edit] from the [Secondary] menu. If your structure contains less than 18 (Atari ST) or less than 21 (Apple Macintosh) stems you will enter the editor without any further ado. If however the structure is too big the box shown below will appear. It enables you to edit a big structure by working on a smaller part. +-------------------------------------+ | Structure too big to fit in editor: | | | | Number of rows = 28 (*1) | | | | Max. number of rows is 18 (*2) | | | | Edit which part ? | | | | Edit row ___ to ___ | | | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------- | +-------------------------------------+ You enter the first and the last stems of the part of the structure that you want to edit at the fields marked by _ . For example, if you want to edit the first five stems of a structure which consists of 30 stems, type 1 in the first field, and 5 at the second. When you have entered these values, press [OK]. ------------------------------------------------------------ (*1) example (*2) Atari ST example: Maximum number of rows for Mac is 21 - 87 - Reference Guide STAR Page 7-21 7 The [Secondary] menu 29 June 1990 If you don't want to edit the structure, press [Cancel]. ________ ______ Possible errors: If you enter an illegal range in the box above or enter other characters than numbers, the box appears again. In fact the box keeps appearing until you enter a correct range. - 88 - Reference Guide STAR Page 7-22 7 The [Secondary] menu 29 June 1990 ___ ______ ______ ___ ______ 7.8.2 The Editor Screen And Cursor When you are in the editor, you see a structure like: ------+------+------+------+ 27| 32| 38| 43| CACCCU..AGGGUG 49| 53| 61| 65| GGCAG..CUGCC 1| 7| 66| 72| GCUGAUA..UAUCAGC | | | | | | | | : : : : : : : : : : : : | | | | ------+------+------+------+ The left part of the screen shows the stems of the structure. The four columns are: - Column 1 contains the base number of the most 5' nucleotide of the stem - Column 2 contains the base number of the 3' end of the 5' stem-half - Column 3 contains the base number of the 5' end of the 3' stem-half - Column 4 contains the base number of the most 3' nucleotide of the stem The right part of the screen shows the nucleotides that base-pair in the particular stem. The .. symbolise the hairpin loop on top of the stem. The cursor of the editor is the blinking dash. At the height of the cursor you can see whether you are working in insert or in overwrite mode. If the cursor is a high dash, you are in overwrite mode. If it is a low dash, you are in insert mode. The meaning of being in insert or overwrite mode is explained in paragraph 7.8.3.3. - 89 - Reference Guide STAR Page 7-23 7 The [Secondary] menu 29 June 1990 ___ ______ ____ 7.8.3 The Editor Keys You can use several keys during editing. We list them in the following paragraphs. ______ _______ ___ _________ 7.8.3.1 Moving Through The Structure To move through the structure, you can use the arrowkeys. As their use is obvious, we don't discuss them further. If you want to move faster through the structure, you can use the [Tab] key. If you press this key, the cursor moves column-by-column trough the structure. Another way to move through the structure is by using the mouse. ________ _ ______ 7.8.3.2 Deleting A Number You can use two different keys to delete a single number; the [Delete] key or the [Backspace] key. If you press [Delete], STAR deletes (erases) the number at cursor position. If you press [Backspace], STAR deletes the number just left of the cursor. You can delete a whole group of numbers by pressing the [Esc] key (Atari) or the [Clear] key (Apple). If you press this key, STAR erases the whole field in which the cursor is positioned. ______ _ ______ 7.8.3.3 Adding A Number You can add a number by simply typing in that number. If you are in 'insert' mode, STAR inserts the number just left of the cursor. If you are in 'overwrite' mode STAR replaces the number at cursor position by the new number. See also paragraph 8.8.2 and paragraph 8.8.3.4. When your structure grows too big during the editing; that is more than 18 stems for Atari users and more than 21 stems for Mac users, STAR is unable to handle your structure anymore in total. So you have to terminate your session and resume editing a smaller part of the structure. STAR warns you with the following box, and you should leave the editor and resume editing with a smaller part of the data. - 90 - Reference Guide STAR Page 7-24 7 The [Secondary] menu 29 June 1990 +----------------------------------+ | The object you are editing | | is grown too big: it doesn't | | fit in the editor anymore | | | | You must either [Exit] or [Quit] | | from the editor. | | | | -------+ +------+ | | | Exit | | Quit | | | +------+ +------- | +----------------------------------+ Pressing the [Exit] button has the same effect as activating the [File/Exit] option from the editormenubar. This means that you leave the editor and fix all the changes that you made. Pressing [Quit] has the same effect as activating the [File/Quit] option from the editor menubar. In this case you will also leave the editor, but you loose all the changes that you made during the edit session. Once you left the editor, you can resume editing though part-wise only; see paragraph 8.8.1. ______ _______ ________ ___ ___________ ____ 7.8.3.4 Toggle Between 'insert' And 'overwrite' Mode You can toggle between 'insert' and 'overwrite' mode using the [Insert] key. You see the cursor change from a high dash to a low dash or vice-versa. When the cursor is a high dash you are in overwrite mode, when it is a low dash you are in insert mode. See also paragraph 8.8.2 and 8.8.3.4. - 91 - Reference Guide STAR Page 7-25 7 The [Secondary] menu 29 June 1990 ___ ______ ____ 7.8.4 The Editor Menu +----------------------------------+ |Desk File Clipboard | +----------------------------------+ When you enter the editor the menubar above appears. As you see, the menubar consists of three menus: [Desk],[File] and [Clipboard]. The [Desk] menu is not really a STAR menu. It contains the desk-accessories that were in the System-file of the diskette you used to start the computer. In the Atari version it also contains the [About STAR] option. We discuss this option in paragraph 5.2. From now on we ignore the [Desk] menu. However, we discuss the other two menus in the following paragraphs. ___ ______ ____ 7.8.4.1 The [File] Menu +------------------+---------------+ |Desk |File | Clipboard | +-----------| +-+-------------+ | Replace| | Glue | |--------| | Exit | | Quit | +--------+ The first two options in the [File] menu, [Replace] and [Glue] are miscellanious editor-options. You can use the last two options to leave the editor and return to the main menu. We fully discuss these options in the following paragraphs. ___ _________ ______ 7.8.4.1.1 The [Replace] Option You can use the [Replace] option only while you are editing a sequence. See paragraph 6.6.4.1.1 ___ ______ ______ 7.8.4.1.2 The [Glue] Option The [Glue] option enables you to add stems to the structure by pointing in the sequence to the single-stranded regions that basepair. When you activate this menu the sequence appears on screen in the - 92 - Reference Guide STAR Page 7-26 7 The [Secondary] menu 29 June 1990 following way: 5 10 15 20 25 30 35 40 45 GCUGA UAUAG UUGGU AGAGC CGAGC CUUGG UAAGG GUGAG GUCGG 50 CAGUU CCAAU CUGCC UAUCA GCACC A 100 Note that this is the same view as presented in paragraph 6.5. Now you can select the first stemhalf by dragging the mouse over the sequence. Dragging means that you point the mouse at the first nucleotide, push the mousebutton and move the mouse to the last nucleotide. During this process you must keep pressing the mousebutton. After releasing the mousebutton, that part will be selected. This is indicated by 'reversed video': white letters on a black background. Next you select the second stemhalf the same way. When you selected both stemhalves (both single-stranded parts that will basepair) the box shown below appears. With this box you can choose whether to add the stem to the structure or not. +------------------------+ | Entered stem: | | | | 7 17 55 65 (*1) | | | | Add it to structure ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------+ When you want to add that stem to your structure, press [OK], else press [Cancel]. Note that STAR does not check if the stem you entered is correct. So STAR will not notify you if both stem- halves have an unequal length. Now another box appears. With this box you can decide whether to enter another stem using the [Glue] option or not. +------------------------+ | Enter another stem ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------+ If you don't want to enter another stem using the [Glue] option, press [CANCEL]. Then you return to the main editor menu. Else press [OK] and the process starts all over again. ------------------------------------------------------------ (*1) example stem - 93 - Reference Guide STAR Page 7-27 7 The [Secondary] menu 29 June 1990 ___ ______ ______ 7.8.4.1.3 The [Exit] Option You can use the [Exit] option to leave the editor and return to the main menu. All changes that you have made during the edit session will be fixed. If you don't want this, you can use the option that we discuss in the next paragraph: [Quit]. STAR doesn't calculate the energy values of the entered stems. STAR will set them to 0. ________ _______ Possible errors: When the editing results in a 'stem' which doesn't contain four values, the box shown below appears. STAR warns you with this box that such a stem is not allowed. +----------------------+ | ERROR: | | Row nr 3: | | 7 66 72 *) | | is not allowed. | | It will be discarded | | +------+ | | | OK | | | +------+ | +----------------------+ *) example wrong stem All you can do is press [OK]. STAR will not add the stem to the structure. When your editing creates 'stems' with unequal lengths of the stemhalves, the box shown below appears. STAR warns you with this box that such stems are not allowed. +-----------------------------+ | ERROR.Stems not allowed: | | 5 10 14 20 *) | | (unequal length stemhalves) | | They will be discarded. | | +------+ | | | OK | | | +------+ | +-----------------------------+ *) example wrong stem All you can do is press [OK]. STAR will not add the stem to the structure. When your editing results in a stem which overlaps with an other stem, the box shown below appears. STAR warns you with this box that such astem is not allowed. - 94 - Reference Guide STAR Page 7-28 7 The [Secondary] menu 29 June 1990 +-----------------------------+ | ERROR.Stem not allowed: | | 5 10 14 20 *) | | (overlap with other stem) | | It will be discarded | | +------+ | | | OK | | | +------+ | +-----------------------------+ *) example wrong stem All you can do is press [OK]. STAR will not add the stem to the structure. ___ ______ ______ 7.8.4.1.4 The [Quit] Option Like the previously discussed option, you can use the [Quit] option to leave the editor and return to the main menu. However when you use this option, the changes you made during the editing ___ will not be fixed. This means that you lose all changes that you have made during the edit session! That is why after activating this option the warning-box as shown below appears. With this box STAR asks you to confirm the quitting from the editor. +--------------------------+ | You are about to QUIT | | from the editor | | ALL CHANGES WILL BE LOST!| | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------------+ If you press [OK], you leave the editor without saving the changes. If you press [Cancel] you don't leave the editor and return to the main editor menu. - 95 - Reference Guide STAR Page 7-29 7 The [Secondary] menu 29 June 1990 ___ ___________ ____ 7.8.4.2 The [Clipboard] Menu +----------------------------------+ |Desk File |Clipboard | +-----------------------| ++ | Cut | | Copy | | Paste | |---------| | Unblock | +---------+ The [Clipboard] menu contains all options that use the clipboard. You are probably familiar with the use of it. If you are not, don't despair as we discuss the basics of using the clipboard below. We discuss the options in the next paragraphs. The clipboard is a piece of memory in your computer into which you can move or copy large pieces of your structure. The way it works is that you first put a structurepart in the clipboard using either the [Cut] or the [Copy] option. Then you can insert it somewhere in your structure using the [Paste] option. In the following paragraphs we discuss these options. ___ _____ ______ 7.8.4.2.1 The [Cut] Option You can use the [Cut] option to transfer stems from the STAR editor to the clipboard. First you select a structurepart by dragging the mouse over the structure. Dragging means that you point the mouse at the first stem, push the mousebutton and move the mouse to the last stem. During this process you must keep pressing the mousebutton. When you now release the mousebutton, STAR selects that part of the structure. You can see this because it is now show in 'reversed video': white letters on a black background. Note that you can only select stem-by-stem: it is not possible to select a part of a stem. The [Cut] option puts the selected stems in the clipboard, and remove them from the editor. If you don't want to erase the selected stems you use the following option: [Copy] - 96 - Reference Guide STAR Page 7-30 7 The [Secondary] menu 29 June 1990 ___ ______ ______ 7.8.4.2.2 The [Copy] Option Similar to the [Cut] option, you can use the [Copy] option to transfer stems from the STAR editor to the clipboard. First you must select a structurepart by dragging the mouse over the structure. If you are not familiar with the term 'dragging' see the previous paragraph, as we explained it there. Note that you can only select stem-by-stem: it is not possible to select a part of a stem. The [Copy] option places a copy of the selected stems in the clipboard. So STAR will not remove the selected stems. If you do want to remove them, see the previous paragraph about the [Cut] option. ___ _______ ______ 7.8.4.2.3 The [Paste] Option You can use the [Paste] option to transfer stems from the clip-board to the STAR editor. STAR doesn't erase the stems in the clipboard, so you can [Paste] the same stems several times. When your structure grows longer than 18 stems for Atari users or more than 21 stems for Apple users, STAR is unable to handle your structure in total anymore. So you must terminate your session and resume editing a smaller part of the structure. STAR warns you with the box that we explained in paragraph 8.8.3.3. ___ _________ ______ 7.8.4.2.4 The [Unblock] Option You can use the [Unblock] option to erase the current block-definition. This is indicated by the stems which STAR shows in normal video again (black letters on a white background). Other ways to erase the blockdefinition are to press the mousebutton once, or to press any key. - 97 - Reference Guide STAR Page 7-31 7 The [Secondary] menu 29 June 1990 _______ 7.9 [PRINT] You can use the [Print] option to make a hard-copy (a print) of your structure. Naturally you can only activate this option if you have a structure in your computer's memory. When you activate this option one of the boxes shown below appear, depending on whether your computer is an Atari or an Apple. With this box you can specify where you want to print to and enter the size of you printer paper. +-------------------------+ --------------------------+ | Print structure as | | Print structure as | | | | | | -------+ ------+ -----+ | | | | |Table | |Span | |Lego| | | o Table o Span | | +------- +------ +----- | | | | | | | | ---------+ | | | | |Mountain| | | o Lego o Mountain | | +--------- | | | | | | | | -------+ +--------+ | | -------+ +--------+ | | | OK | | Cancel | | | | OK | | Cancel | | | +------+ +--------- | | +------+ +--------- | +-------------------------- +-------------------------+ Atari box Apple box By this box, you can select the way to print the structure. These ways are the same as described by the [View] option; see paragraph 8.7 When you have chosen the way to print the structure one of the following boxes appear, depending on whether your computer is an Atari or an Apple: +------------------------------+ ----------------------------+ | PRINT OPTIONS | | PRINT OPTIONS | | | | | | Page length __ | | Printer o Image o Deskjet | | Page width __ | | | | Top margin __ | | Page length __ | | Bottom margin __ | | Page width __ | | | | Top margin __ | | +-------+ --------+ | | Bottom margin __ | | Print to:|Printer| | File | | | | | +-------- +-------- | | Print to: o Printer o File| | | | | | -------+ +------+ | | -------+ +------+ | | |Cancel| | OK | | | |Cancel| | OK | | | +------+ +------- | | +------+ +------- | +------------------------------- +---------------------------+ Atari box Apple box - 98 - Reference Guide STAR Page 7-32 7 The [Secondary] menu 29 June 1990 In the four fields marked by __, you must enter the length and width of the paper you use, the top margin and the bottom margin. The top margin is the blank part above the text on a page. The bottom margin is the blank part below the text on a page. You must enter all these settings in number of characters. For instance, if you use paper 71 characters long, enter '71' at the first field. For a standard A4 paper format, you must enter '66' for the pagelength, and '80' for the page width. For standard continuous forms paper, enter '71' for the pagelength and '80' for the pagewidth. We advise for both top and bottom margin a length of 2 characters. If you use a MacIntosh you must specify the type of your printer. If your printer is an Apple ImageWriter, you must select the [Image] button. If your printer is a HP Deskjet+, you must select the [Deskjet] button. HP Deskjet+ users should also set DIP-switch number 5 from the rightmost DIP-switch block to on. This DIP-switch block is positioned at the front of the printer. Both Atari and Apple users can specify whether they want to print to a printer or to a file. Printing to a printer means that STAR outputs directly to a printer. In this case a printer must be attached to your computer and it must be switched on and on-line. If you don't have a printer attached, but a printer attached to another computer is available, you can print to file. In this case STAR asks you to enter a filename using a file-selector. If you are not familiar with the use of a file-selector, please consult the manual that was delivered with your computer. Now STAR will make a printerfile for you. ________ ______ Possible errors: If you specify you want to print to a printer while there is no printer attached to your computer, you can not use STAR for about 30 seconds. - 99 - CHAPTER 8 ___ _____________ ____ THE [ENERGYRULES] MENU +-----------------------------------------------------------+ |Desk Files Primary Secondary |Energyrules| +-----------------------------------------------| | | Help | |-----------| | Open | | Save as...| | Save | |-----------| | View | | Edit | |-----------| | Print | +-----------+ In this chapter, we fully explain all options in the [Energyrules] menu. - 100 - Reference Guide STAR Page 8-2 8 The [ENERGYRULES] menu 29 June 1990 ______ 8.1 [HELP] The [Help] option displays brief information about the options in this menu. The main purpose of this option is to give you some help at your fingertips, without having to consult the manual. Now don't throw away this manual as this option is only a brief reminder. When you press the mousebutton or a key (Apple version) or the [OK] button (Atari version), you leave this option and return to the main menu. - 101 - Reference Guide STAR Page 8-3 8 The [ENERGYRULES] menu 29 June 1990 ______ 8.2 [OPEN] The [Open] option enables you to open a file with energy rules from disk. This means that STAR will transfer a copy of a set of energy rules from disk to the computer. Then you can use it with STAR. As STAR always has a default set of energy rules, the warning box shown below always appears before you can open an energy rules file. With this box STAR asks you to confirm the opening of a (new) energy rules file. +--------------------------------+ | Opening a energy rules file | | will overwrite your current | | energy rules! | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------------------+ If you press [OK], STAR erases the energy rules that are in the computer and you are able to open another energy rules file. This means you lose the energy rules in the computer! So, please _____ be sure that you have saved your current set of energy rules _______ (using the options [Save] or [Save as...]) or that it is useless before you press [OK]. If you are not sure press [Cancel]. Then STAR erases nothing and you return to the main menu. When you have pressed [OK] a file-selector will appear by which you can select the energy rules file you want to open. If you are not familiar with the use of a file-selector, consult the manual that was delivered with your computer. The energy rules are very specific for STAR. Therefore, you can only open energy rules files that you once saved with STAR. If you try to open a file that is not an energy rules file (not possible on Apple), the following box will appear: +-----------------------+ | This file is not an | | energy rules file! | | | | +------+ | | | OK | | | +------+ | +-----------------------+ All you can do is press [OK], and you will return to the main menu. ________ _______ Possible errors: - 102 - Reference Guide STAR Page 8-4 8 The [ENERGYRULES] menu 29 June 1990 WS-FULL A very obvious error is when you want to open an energy rules file that is too big for the memory of your computer. This results in a 'WS-FULL' error. Unfortunately you can do nothing else than start STAR all over again on a computer with sufficient memory. - 103 - Reference Guide STAR Page 8-5 8 The [ENERGYRULES] menu 29 June 1990 _____ ______ 8.3 [SAVE AS...] The [Save as...] option enables you to save your current set of energy rules for later use. This means that STAR transfers a copy of the set in the computer's memory to disk. You must enter the name of the file where you want to save the energy rules to. When you activate this option a file-selector appears by which you can enter the filename under which STAR will save the energy rules. If you are not familiar with the use of a file-selector, consult the manual that was delivered with your computer. If you enter a filename that already exists, the box shown below appears. With this box STAR asks you if you want to overwrite the file. +------------------------------+ | File 'filename' | | already exists. Overwrite ? | | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +------------------------------+ If you press [OK], STAR saves the energy rules to disk. It overwrites the energy rules on disk. Otherwise press [Cancel]. Then STAR overwrites nothing and you return to the main menu. - 104 - Reference Guide STAR Page 8-6 8 The [ENERGYRULES] menu 29 June 1990 ______ 8.4 [SAVE] The [Save] option enables you to save your current set of energy rules for later use. This means that STAR transfers a copy of the set in the computer's memory to disk. The difference with the option [Save as...] is that STAR saves the energy rules to a file with the same name as you opened. That is why you must have opened an energy rules file (option [Open]) before you can activate this option. Furthermore, STAR replaces the energy rules on disk by the energy rules that you saved without warning, so be careful with this option: be sure that the energy rules on disk are no longer useful before you use this option. - 105 - Reference Guide STAR Page 8-7 8 The [ENERGYRULES] menu 29 June 1990 ______ 8.5 [VIEW] You can use the [View] option to view the energy rules that are in the computer's memory. Because there are several energy rules, you must first select the type of energy rules you want to view. You can do that using one of the following boxes that will appear immediately after you activate this option. The type of box depends on whether your computer is an Apple or an Atari: +---------------------------------------+ | Choose name of energy rules to display| | | | o Stacking o Interior loop | | | | o Bulge o Hairpin | | | | o Deep groove o Shallow groove | | | | o Backbone | | | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------- | +---------------------------------------+ Apple box +----------------------------------------+ | Choose name of energy rules to display | | | |--------------+ +-------------+ | ||Stacking | |interior loop| | |+-------------+ +-------------- | |--------------+ +-------------+ | ||Bulge | |Hairpin | | |+-------------+ +-------------- | |--------------+ +-------------+ | ||deep groove | |shallowgroove| | |+-------------+ +-------------- | |--------------+ | ||Backbone | | |+-------------+ | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------+ | +----------------------------------------+ Atari box You must use the so-called radiobuttons to select the type of energy rules. - 106 - Reference Guide STAR Page 8-8 8 The [ENERGYRULES] menu 29 June 1990 When you press the [OK] button, STAR will display the energy rules. When you press [Cancel], nothing will happen and you will return to the main menu. As you see there are seven types of energy rules. We explain them now. _________ 1. Stacking: the energy values for all possible stackings of basepairs. ________ _____ 2. Interior loop: the energy values for interior loops. ______ 3. Bulge: the energy values for bulge loops. ________ 4. Hairpin: the energy values for hairpin loops. ____ _______ 5. Deep groove: one of the energy values for p-knots _______ _______ 6. Shallow groove: one of the energy values for p-knots. In a p-knot one loop crosses the deep groove. The other crosses the shallow groove. When you follow the RNA strand in a p-knot from the 3'side, the first loop you meet crosses the shallow-groove. When you follow the RNA strand from the 5'side, the first loop you meet crosses the deep groove. This is illustrated in figure below. 5' : +---------+ : | | | 3'>-------- --------3-+ 5' 3'-------------------+ 5' -----------------+ 5' >------+ +-------3--+-- 3' | | | : +---------+ : 3' shallow groove deep groove ________ 7. Backbone: the energy values for a-knots. You can see an a-knot as an ordinary stem-loop structure of which the free 3'end or the free 5' end crosses the backbone of the stem and basepairs with the loop. You see this in the figure shown below which shows the formation of a 3' a-knot. In this figure, 3 nucleotides cross the backbone while spanning 9 base pairs. - 107 - Reference Guide STAR Page 8-9 8 The [ENERGYRULES] menu 29 June 1990 ++UAGCACGC+3' +-+U-+A-+G-+ | | 3' | UGAC-? ------+> UGAC-?+CGCAC 5'-+ACUG-3+GCGUG 5'-+ACUG-3+GCGUG | | | | ++C+G-Y @+C+G-+ Stem-loop 3' a-knot In the following paragraphs we discuss the display of each type of energy rules. _______ ________ ______ _____ 8.5.1 [View]: Stacking Energy Rules The stacking energy rules include the energy values that follow from the stacking of basepairs. STAR displays them in a table, as shown below. __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ AA AU AG AC UA UU UG UC GA GU GG GC CA CU CG CC __ AA 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ AU 20-12 20 20-12 20 -3 20 20 -3 20-21 20 20-21 20 __ AG 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ AC 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ UA 20-18 20 20-12 20 -3 20 20 -3 20-21 20 20-21 20 __ UU 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ ___ UG 20 -3 20 20 -3 20 -3 20 20 -3 20-13 20 20-13 20 <=== __ UC 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ GA 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ GU 20 -3 20 20 -3 20 -3 20 20 -3 20-13 20 20-13 20 __ GG 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ GC 20-21 20 20-21 20-13 20 20-13 20-48 20 20-30 20 __ CA 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ CU 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 __ CG 20-21 20 20-21 20-13 20 20-13 20-43 20 20-48 20 __ CC 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 In this table, you find the energy of a particular stack by selecting the row with the bottom base-pair in the left column and the top base-pair in the first line. The intersection is the Gibbs Free Energy of the stack in 0.1kcal/mol. For example if a GC basepair stacks upon a UG basepair, the Gibbs Free Energy is -13 0.1kcal/mol. See the underscored number in above table. Note that the energy value of a stack is always the same as its mirror-image. For example the energy of a GC-UG is equal to the value of a GU-CG stack, as GU-CG is the mirror-image of GC-UG. This explains the energy of a stack of two basepairs. To calculate the energy of a stack of more basepairs we give the - 108 - Reference Guide STAR Page 8-10 8 The [ENERGYRULES] menu 29 June 1990 following example: GC CG -43 AU -21 GU -3 ----- + -67 So you start at the top of the stack and work your way downwards until the stack has ended. For each stack of two basepairs, you search the energy in the table. Finally you add these values to get the Gibbs Free Energy of the stack. _______ ________ ____ 8.5.2 [View]: Interior Loop When you view the energy rules of interior loops, you get a five- column table like: 2 1 9 17 24 3 10 18 26 34 __ 4 16 25 33 40 <=== 5 22 30 38 46 7 29 38 46 53 The first column gives the length of the interior loop. The next four columns give the Gibbs Free Energy in 0.1kcal/mol for four different combinations of closing basepairs of the interior loop. The second column gives the energy values for closing pair GC-GC. The third column gives the energy values for closing pair GC-AU. The fourth column gives the energy values for closing pairs AU-AU and GC-other than GC or AU. The last column gives the energy value for closing pairs AU-other than GC or AU. If for example you want to know the energy value for an interior loop of length 4 and with closing pair AU-AG, you must look in the last column in the row with 4 in the first column. In this example the value is 40 0.1kcal/mol. When the values for a certain length are missing, STAR uses the most nearby lower value. In this example the values for an interior loop 6 nucleotides long are 22 30 38 46. _______ _____ ____ 8.5.3 [View]: Bulge Loop When you view the energy rules of a bulge loop, you see a two-column table, like: - 109 - Reference Guide STAR Page 8-11 8 The [ENERGYRULES] menu 29 June 1990 0 40 1 30 __ 2 46 <=== 3 55 4 62 6 71 In the first column specifies the length of the bulge loops. The second column specifies the Gibbs Free Energy of the bulge loops. If you want to know for example the energy of a bulge loop 2 nucleotides long, you look in the second column where the first column is 2. In this example the energy value is 46 0.1kcal/mol. When a value is not defined, STAR uses the most nearby lower value. In this example the energy of a bulge loop length of 5 nucleotides is 62 0.1kcal/mol. _______ _______ ____ 8.5.4 [View]: Hairpin Loop When you view the energy rules of a hairpin loop, you see a two- column table like: 1 999 5 70 __ 6 45 <=== 7 41 8 43 In the first column STAR presents the length of the hairpin loops. The second column specifies the Gibbs Free Energy of the hairpin-loops. For example if you want to know the energy of a 6 nucleotide long hairpin loop, you look in the second column where the first column is 6. In this example the energy value is 45 0.1kcal/mol. When a value is not defined, STAR uses the most nearby lower value. In this example the energy of a hairpin loop with a length of 4 nucleotides is 999. _______ ____ ______ 8.5.5 [View]: Deep Groove When you view the energy rules of a deep groove, you see a table like: - 110 - Reference Guide STAR Page 8-12 8 The [ENERGYRULES] menu 29 June 1990 0 1 2 15 16 2 999 999 999 999 3 999 42 42 999 __ 4 999 42 42 999 <=== 8 42 42 42 999 9 42 42 42 999 10 999 999 999 999 The top row specifies the number of nucleotides that cross the deep groove. The left column specifies the number of base pairs to be crossed. For example you want to know the energy value of a connecting loop of 2 nucleotides crossing the deep groove over 4 base pairs. The energy value is then 42 0.1kcal/mol. _______ _______ ______ 8.5.6 [View]: Shallow Groove When you view the energy rules of a connecting loop spanning a shallow groove, you see a table like: 0 1 2 15 16 2 999 999 999 999 3 999 42 42 999 __ 4 999 42 42 999 <=== 8 42 42 42 999 9 42 42 42 999 10 999 999 999 999 The top row specifies the number of nucleotides that cross the shallow groove. The left column specifies the number of basepairs to be crossed. For example you want to know the energy value of a connecting loop of 2 nucleotides crossing the shallow groove over 4 base pairs. The energy value is then 42 0.1kcal/mol _______ ________ 8.5.7 [View]: Backbone When you view the energy rules of a backbone, you get a table like: 0 1 2 1 999 999 2 999 999 The top row specifies the number of nucleotides that cross the backbone. The left column specifies the number of base pairs to be crossed. - 111 - Reference Guide STAR Page 8-13 8 The [ENERGYRULES] menu 29 June 1990 ______ 8.6 [EDIT] The [Edit] option enables you to change the default energy rules if you are not satisfied with them. We will explain the editor in the following paragraphs. ________ ___ ______ 8.6.1 Entering The Editor You enter the editor by activating the option [Edit] from the [Energyrules] menu. Then one of the following boxes appears, depending if your computer is an Atari or an Apple: +---------------------------------------+ | Choose name of energy rules to change | | | | o Stacking o Interior loop | | | | o Bulge o Hairpin | | | | o Deepgroove o Shallow groove | | | | o Backbone | | | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------- | +---------------------------------------+ Apple box +----------------------------------------+ | Choose name of energy rules to change | | | |--------------+ +-------------+ | ||Stacking | |Interior loop| | |+-------------+ +-------------- | |--------------+ +-------------+ | ||Bulge | |Hairpin | | |+-------------+ +-------------- | |--------------+ +-------------+ | ||Deep groove | |Shallowgroove| | |+-------------+ +-------------- | |--------------+ | ||Backbone | | |+-------------+ | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------+ | +----------------------------------------+ Atari box - 112 - Reference Guide STAR Page 8-14 8 The [ENERGYRULES] menu 29 June 1990 You must use the so called radiobuttons to select the type of energy rules you want to edit. If you don't understand the different types of energy rules, look in paragraph 8.5 as we explained them there. When you press the [OK] button, you will enter the editor; when you press [Cancel], you will return to the main menu. If the energy rules you selected contain less than 18 (Atari ST) or less than 21 (Apple Macintosh) rows you enter the editor without any further ado. If however the table with energy rules is too big the box shown below appears. It enables you to edit a big table with energy-rule by working on a smaller part. +-------------------------------------+ | xxxx (*1) too big to fit in editor: | | | | Number of rows = 28 (*2) | | | | Max. number of rows is 18 (*3) | | | | Edit which part ? | | | | Edit row ___ to ___ | | | | | | -------+ +------+ | | | OK | |Cancel| | | +------+ +------+ | +-------------------------------------+ You must enter the first and the last row of that part of the table you want to edit in the fields marked by ___ . For example, if you want to edit the first 10 rows of a table of 25 rows with energy-rules, type 1 at the first field and 25 at the second. When you have entered these values, press [OK]. If you don't want to edit, press [Cancel]. ________ ______ Possible errors: If you enter an illegal range in the box above or enter other characters than numbers, the box appears again. In fact the box keeps appearing until you enter a correct range. For some energy rules: shallow groove, deep groove and Backbone, there is also a maximum number of columns you can edit at a time. You can edit not more then 10 columns at once. If your energy rules contain more that 10 columns, a box like shown above appears. With this box, STAR lets you enter the first and last column you want to edit. For example, if you want to edit the first 4 columns of a table, you enter 1 in the first field and 4 ------------------------------------------------------------ (*1) type of energy rules you want to edit. (*2) example. (*3) Atari ST example. - 113 - Reference Guide STAR Page 8-15 8 The [ENERGYRULES] menu 29 June 1990 at the second. Remember that if you add extra rows to the tablepart you edit, STAR will add zeros to the rows that you do not edit. For example, you edit the middle two rows of the table below: 1 2 3 4 5 6 7 8 And you add the values 99 to the middle two columns during the editing. After the editing the total table will be: 1 2 3 4 5 6 7 8 0 99 99 0 ___ ______ ______ ___ ______ 8.6.2 The Editor Screen And Cursor When you have entered the editor, you see the energy rules on screen. There are 7 different types of energy rules and the editorscreen differs for all of them. In the next paragraphs we explain the editorscreen for each type of energy rules. The cursor of the editor is the blinking dash. At the height of the cursor you can see whether you are working in insert or in overwrite mode. If the cursor is a blinking high dash, you are in overwrite mode. If it is a low dash, you are in insert mode. The meaning of being in insert or overwrite mode is explained in paragraph 8.6.3.3. ___ ______ ______ _____ _______ ________ ______ _____ 8.6.2.1 The Editor Screen While Editing Stacking Energy Rules When you are editing Stacking Energy rules, you see a table like: - 114 - Reference Guide STAR Page 8-16 8 The [ENERGYRULES] menu 29 June 1990 AA| AU| AG| AC| UA|....UC| GA| GU| GG| GC| CA| CU| CG| CC| --+---+---+---+---+....--+---+---+---+---+---+---+---+---+ 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|AA 20|-12| 20| 20|-12|....20| 20| -3| 20|-21| 20| 20|-21| 20|AU 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|AG 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|AC 20|-18| 20| 20|-12|....20| 20| -3| 20|-21| 20| 20|-21| 20|UA 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|UU 20| -3| 20| 20| -3|....20| 20| -3| 20|-13| 20| 20|-13| 20|UG 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|UC 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|GA 20| -3| 20| 20| -3|....20| 20| -3| 20|-13| 20| 20|-13| 20|GU 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|GG 20|-21| 20| 20|-21|....20| 20|-13| 20|-48| 20| 20|-30| 20|GC 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|CA 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|CU 20|-21| 20| 20|-21|....20| 20|-13| 20|-43| 20| 20|-48| 20|CG 20| 20| 20| 20| 20|....20| 20| 20| 20| 20| 20| 20| 20| 20|CC  | | | | |.... | | | | | | | | |  | | | | |.... | | | | | | | | | As you can see, the stacking energy rules are collected in a table. This table is basically the same as the one discussed in paragraph 8.5.1. by the [View] option. The main difference is that here STAR separates the columns by | and that STAR shows the top basepairings in the right column of the table instead of in the left. ___ ____________ _____ _______ ________ ____ ______ 8.6.2.2 The Editorscreen While Editing Interior Loop Energy _____ Rules When you are editing interior loop energy rules, you see a table like: ------+-----+-----+-----+-----+ 2| 1| 9| 17| 24|Col 1:length of interior loop 3| 10| 18| 26| 34| 4| 16| 25| 33| 40|Col 2:closing pair GC-GC 5| 22| 30| 38| 46| 7| 29| 38| 46| 53|Col 3:closing pair GC-AU | | | | | | | | | |Col 4:closing pair AU-AU, : : : : : GC-other than GC or AU : : : : : : : : : :Col 5:closing pair AU-other : : : : : than GC or AU | | | | | ------+-----+-----+-----+-----+ As you see, STAR collects the interior loop energy rules in a table. This table is basically the same as the one discussed in - 115 - Reference Guide STAR Page 8-17 8 The [ENERGYRULES] menu 29 June 1990 paragraph 8.5.2. by the [View] option. The difference is that here STAR separates the columns by 3. ___ ____________ _____ _______ _____ ____ ______ _____ 8.6.2.3 The Editorscreen While Editing Bulge Loop Energy Rules When you are editing the energy rules of a bulge loop, you see a two-column table like: -------+------+ 0| 40| Col 1:length of bulge loop 1| 30| 2| 46| Col 2:energy of bulge loop 3| 55| 4| 62| 6| 71| | | : : : : | | -------+------+ This table is the same as the one discussed by the [View] option in paragraph 8.5.3, only here STAR separates the columns by |. ___ ____________ _____ _______ _______ ____ ______ _____ 8.6.2.4 The Editorscreen While Editing Hairpin Loop Energy Rules When you are editing the energy rules of a bulge loop, you see a two-column table, like: -------+-------+ 1| 999| Col 1:length of hairpin loop 5| 70| 6| 45| Col 2:energy of hairpin loop 7| 41| 8| 43| | | : : : : | | -------+-------+ This table is the same as the one discussed by the [View] option in paragraph 8.5.4, only here STAR separates the columns by |. - 116 - Reference Guide STAR Page 8-18 8 The [ENERGYRULES] menu 29 June 1990 ___ ____________ _____ _______ ____ ______ ______ _____ 8.6.2.5 The Editorscreen While Editing Deep Groove Energy Rules When you are editing the energy rules of a connecting loop spanning a deep groove, you see a table like: -DB----+----+----+----+----+----+----+----+ 0| 1| 2| 15| 16| 0| 0| 0| 0|Hor:# of nucl. 2| 999| 999| 999| 999| 0| 0| 0| 0| that cross 3| 999| 42| 42| 999| 0| 0| 0| 0| the groove 4| 999| 42| 42| 999| 0| 0| 0| 0| 8| 42| 42| 42| 999| 0| 0| 0| 0|Ver:# of base pairs 9| 42| 42| 42| 999| 0| 0| 0| 0| to be 10| 999| 999| 999| 999| 0| 0| 0| 0| crossed  | | | | | | | | | : : : : : : : : : : : : : : : : : :  | | | | | | | | | -DA----+----+----+----+----+----+----+----+ This table is basically the same as the one discussed by the [View] option in paragraph 8.5.5., the differences are that here STAR separates the columns by 3 and that STAR shows some extra columns, filled with 0. The purpose of these columns is to make you able to add new columns to the table with energy-rule. ___ ____________ _____ _______ _______ ______ ______ 8.6.2.6 The Editorscreen While Editing Shallow Groove Energy _____ Rules When you are editing the energy rules of a connecting loop spanning a shallow groove, you see a table like: -DB----+----+----+----+----+----+----+----+ 0| 1| 2| 15| 16| 0| 0| 0| 0|Hor:# of nucl. 2| 999| 999| 999| 999| 0| 0| 0| 0| that cross 3| 999| 999| 42| 999| 0| 0| 0| 0| the groove 4| 999| 42| 42| 999| 0| 0| 0| 0| 8| 999| 42| 42| 999| 0| 0| 0| 0|Ver:# of base pairs 9| 999| 42| 42| 999| 0| 0| 0| 0| to be 10| 999| 999| 999| 999| 0| 0| 0| 0| crossed  | | | | | | | | | : : : : : : : : : : : : : : : : : :  | | | | | | | | | -DA----+----+----+----+----+----+----+----+ This table is basically the same as the one discussed by the [View] option in paragraph 7.5.6., the differences are that here STAR separates the columns by 3 and that STAR shows some extra columns filled with 0. The purpose of these columns is to make you able to add new columns to your energy rules. - 117 - Reference Guide STAR Page 8-19 8 The [ENERGYRULES] menu 29 June 1990 ___ ____________ _____ _______ ________ ______ _____ 8.6.2.7 The Editorscreen While Editing Backbone Energy Rules When you are editing the energy rules of a connecting loop spanning a backbone, you see a table like: --------+----+----+----+----+----+----+ 0| 1| 2| 0| 0| 0| 0| Hor:# of nucl. that 1| 999| 999| 0| 0| 0| 0| cross the backbone 2| 999| 999| 0| 0| 0| 0|  | | | | | | | Ver:# of base pairs : : : : : : : to be crossed : : : : : : :  | | | | | | | --------+----+----+----+----+----+----+ This table is basically the same as the one discussed by the [View] option in paragraph 8.6.7., the differences are that here STAR separates the columns by 3 and that STAR shows some extra columns filled with 0. The purpose of these columns is to make you able to add new columns to your energy rules. ___ ______ ____ 8.6.3 The Editor Keys There are several keys you can use during editing. We list them in the following paragraphs. ______ _______ ___ ______ _____ 8.6.3.1 Moving Through The Energy Rules To move through the energy rules, you can use the arrowkeys. As their use is obvious, we don't discuss them further. If you want to move faster through the energy rules, you can use the [Tab] key. If you press this key, the cursor will move column-by-column trough the energy rules. Another way of moving through the structure is by using the mouse. ________ _ ______ 8.6.3.2 Deleting A Number You can use two different keys to delete a number; the [Delete] key or the [Backspace] key. If you press [Delete], STAR deletes (erases) the number at cursor position. If you press [Backspace] STAR deletes the number just left of the cursor. - 118 - Reference Guide STAR Page 8-20 8 The [ENERGYRULES] menu 29 June 1990 You can delete a whole group of numbers by pressing the [Esc] key (Atari) or the [Clear] key (Apple). If you press this key, STAR erases the whole field in which the cursor is positioned. When you are editing the Stacking energy rules, the energy value corresponding with the mirror-image of the stack you are editing will change too. For example, you are editing the energy value for the stack AU-CG. When you change this energy value, the energy value for the stack GC-UA will change too. See also paragraph 8.5.1. ______ _ ______ 8.6.3.3 Adding A Number You can add a number by simply typing in that number. If you are in 'insert' mode, STAR inserts the number just left of the cursor. If you are in 'overwrite' mode STAR, replaces the number at cursor position by the new number. When you are editing the Stacking energy rules, the energy-rule of the stack corresponding with the mirror-image of the stack you are editing will change too. For example, you are editing the energy value for the stack AU-CG. When you change this value, the value for the stack GC-UA will change also. See also paragraph 8.5.1. If your table with energy rules grows too big during the editing; that is more than 18 rows for Atari users and more than 21 rows for Apple users, the editor is unable to handle your energy-rule in total anymore. So you must terminate your session and resume editing of parts of the energy rules. STAR warns you with the following box, and you should leave the editor and resume editing with a smaller part of the data. +----------------------------------+ | The object you are editing | | is grown too big: it doesn't | | fit in the editor anymore | | | | You must either [Exit] or [Quit] | | from the editor. | | | | -------+ +------+ | | | Exit | | Quit | | | +------+ +------- | +----------------------------------+ Pressing the [Exit] button has the same effect as activating the [Edit/Exit] option from the editormenubar. This means that you leave the editor and fix all the changes that you made. Pressing [Quit] has the same effect as activating the [Edit/quit] option from the editor menubar. In this case you will also leave - 119 - Reference Guide STAR Page 8-21 8 The [ENERGYRULES] menu 29 June 1990 the editor, but you loose all the changes that you made during the edit session. Once you left the editor, you can resume editing though part-wise only; see paragraph 7.8.1. ______ _______ ________ ___ ___________ ____ 8.6.3.4 Toggle Between 'insert' And 'overwrite' Mode You can toggle between 'insert' and 'overwrite' mode using the [Insert] key. You can see the cursor change from a high dash to a low dash or vice-versa. When the cursor is a high dash you are in overwrite mode, when it is a low dash you are in insert mode. See also paragraphs 8.6.2 and 8.6.3.3. - 120 - Reference Guide STAR Page 8-22 8 The [ENERGYRULES] menu 29 June 1990 ___ ______ ____ 8.6.4 The Editor Menu +----------------------------------+ |Desk File Clipboard | +----------------------------------+ When you enter the editor the menubar above appears. As you see, the menubar consists of three menu's: [Desk],[File] and [Clipboard]. The [Desk] menu is not really a STAR menu. It contains the desk- accessories that were in the System-file of the diskette you used to start the computer. In the Atari version it also contains the [About STAR...] option. We discuss this option in paragraph 5.2. From now on we ignore the [Desk] menu. However, we discuss the other two menus in the following paragraphs. ___ ______ ____ 8.6.4.1 The [File] Menu +------------------+---------------+ |Desk |File | Clipboard | +-----------| +-+-------------+ | Replace| | Glue | |--------| | Exit | | Quit | +--------+ The first two options in the [File] menu, [Replace] and [Glue] are miscellaneous editor-options. You can use the last two options to leave the editor and return to the main menu. We fully discuss these options in the following paragraphs. _________ 8.6.4.1.1 [Replace] You can only use this option while you are editing a sequence. See paragraph 6.6.4.1.1 ___ ______ ______ 8.6.4.1.2 The [Glue] Option You can only use this option when you are editing a structure. See paragraph 7.8.4.1.2. - 121 - Reference Guide STAR Page 8-23 8 The [ENERGYRULES] menu 29 June 1990 ___ ______ ______ 8.6.4.1.3 The [Exit] Option You can use the [Exit] option to leave the editor and return to the main menu. All changes that you have made during the edit session will be fixed. If you don't want this, you can use the option that is discussed in the next paragraph: [Quit] ________ _______ Possible errors: If you made a row during editing which doesn't contain enough values, the box shown below appears. With this box STAR warns you that this particular row is not allowed. +----------------------+ | ERROR: | | Row nr 3: | | 7 66 72 *) | | is not allowed. | | It will be discarded | | +------+ | | | OK | | | +------+ | +----------------------+ *) example of wrong row Unfortunately, all you can do is press [OK]. The wrong row will not be incorporated in the energy rules. When you have edited Stacking energy rules, and the size of the table with energy rules is unequal to 16x16 (16 columns and 16 rows), the box shown below appears. With this box, STAR warns you that the size of the Stacking energy rules is not appropriate. +---------------------------+ | The edited stacking rules | | are not of the right size | | They will be discarded | | +------+ | | | OK | | | +------+ | +---------------------------+ Unfortunately, all you can do is press [OK]. STAR discards the edited stacking energy rules. This means that you will lose all changes you made during the edit session. So please make sure not to add or delete rows during the editing of stacking energy rules. When you have edited interior loop, bulge loop, hairpin loop, shallow groove, deep groove or backbone energy rules, and you made double or negative values in the first column, the box shown below appears. With this box, STAR warns you that those values are not allowed. Assume for example that you have edited interior loop energy rules and that you made the following energy values: - 122 - Reference Guide STAR Page 8-24 8 The [ENERGYRULES] menu 29 June 1990 2 1 9 17 24 3 10 18 26 34 2 16 25 33 49 In this example you defined two rows with energy values for loops with a length of 2 nucleotides. This is not allowed, so the box shown below appears: +-----------------------------------------------+ | ERROR. Double or negative values in column 1: | | 2 16 25 33 49 *) | | Rows will be discarded | | +------+ | | | OK | | | +------+ | +-----------------------------------------------+ *) example of wrong row Unfortunately, all you can do is press [OK]. The wrong rows will not be incorporated in the energy rules. When you have edited shallow groove, deep groove or backbone energy rules, and you made double or negative values in the first row, the box shown below appears. With this box STAR warns you that those values are not allowed. Assume for example that you have edited backbone energy rules, and that you made the following energy values: 0 1 2 1 1 999 999 10 2 999 999 20 In this example you defined two columns with energy values for 1 nucleotide crossing the backbone. This is not allowed, so the box shown below appears: +--------------------------------------------+ | ERROR. Double or negative values in row 1: | | 1 *) | | 10 | | 20 | | Columns will be discarded | | +------+ | | | OK | | | +------+ | +--------------------------------------------+ *) example of wrong column Unfortunately, all you can do is to press [OK]. The wrong columns will not be incorporated in the energy rules. - 123 - Reference Guide STAR Page 8-25 8 The [ENERGYRULES] menu 29 June 1990 ___ ______ ______ 8.6.4.1.4 The [Quit] Option Like the previously discussed option, you can use the [Quit] option to leave the editor and return to the main menu. However when you use this option, the changes you made during the editing ___ will not be fixed. This means that you will loose all changes that you made! That is why after activating this option the warning-box shown below appears. With this box STAR asks you to confirm the quitting from the editor. +--------------------------+ | You are about to QUIT | | from the editor | | ALL CHANGES WILL BE LOST!| | | | -------+ +------+ | | |Cancel| | OK | | | +------+ +------- | +--------------------------+ If you press [OK], you leave the editor without saving the changes. If you press [Cancel] you don't leave the editor and return to the main editor menu. - 124 - Reference Guide STAR Page 8-26 8 The [ENERGYRULES] menu 29 June 1990 ___ ___________ ____ 8.6.4.2 The [Clipboard] Menu +----------------------------------+ |Desk File |Clipboard | +-----------------------| ++ | Cut | | Copy | | Paste | |---------| | Unblock | +---------+ The [Clipboard] menu contains all options that use the clipboard. You are probably familiar with the use of it. If you are not, don't despair as we discuss the basics of using the clipboard below. We discuss the options in the next paragraphs. The clipboard is a piece of memory in your computer which you can use to move or copy large pieces of your energy rules. The way it works is that you first put a part of the energy rules in the clipboard using either the [Cut] or the [Copy] option. Then you can insert it somewhere in your energy rules using the [Paste] option. We discuss these options in the following paragraphs. ___ _____ ______ 8.6.4.2.1 The [Cut] Option You can use [Cut] to transfer energy values from the STAR editor to the clipboard. First you select a part of the energy rules by dragging the mouse over the energy values. Dragging means that you point the mouse at the first energy value, push the mousebutton and move the mouse to the last energy value. During this process you must keep pressing the mousebutton. When you now release the mousebutton, STAR selects that part of the energy rules. You can see this because it is now shown in 'reverse video': white letters on a black background. Note that you can only select row-by-row: it is not possible to select a part of a row. The [Cut] option puts the selected energy values in the clipboard, and removes the selected part from the editor. If you don't want to remove that part of the energy rules, you must use the following option: [Copy] - 125 - Reference Guide STAR Page 8-27 8 The [ENERGYRULES] menu 29 June 1990 ___ ______ ______ 8.6.4.2.2 The [Copy] Option As the [Cut] option, you can use the [Copy] option to transfer energy values from the STAR editor to the clipboard. First you must select a part of the energy rules by dragging the mouse over the energy rules. If you are not familiar with the term 'dragging', see the previous paragraph as we explained it there. Note that you can only select row-by-row: it is not possible to select a part of a row. The [Copy] option places a copy of the selected part of the energy rules in the clipboard. So STAR does not remove the selected part of the energy rules. If you do want to remove it, see the previous paragraph about the [Cut] option. ___ _______ ______ 8.6.4.2.3 The [Paste] Option You can use the [Paste] option to transfer energy values from the clipboard to the STAR editor. STAR doesn't erase the energy values in the clipboard, so you can [Paste] the same text several times. When your energy rules table grows longer than 18 rows for Atari users or 21 rows for Apple users, STAR is unable to handle your table with energy rules in total anymore. So you must terminate your session and resume editing a smaller part of the energy rules. STAR warns you with the box explained in paragraph 8.6.3.3. ___ _________ ______ 8.6.4.2.4 The [Unblock] Option You can use the [Unblock] option to erase the current block-definition. You can see this by the text which STAR displays now in normal video again (black letters on a white background). Other ways to erase the blockdefinition are to press the mousebutton once, or to press any key. - 126 - Reference Guide STAR Page 8-28 8 The [ENERGYRULES] menu 29 June 1990 _______ 8.7 [PRINT] You can use the [Print] option to make a hard-copy (a print) of your energy rules. Naturally you can only activate this option if you have energy rules in your computer's memory. When you activate this option one of the following boxes appears, depending on whether your computer is an Atari or an Apple: +------------------------------+ ----------------------------+ | PRINT OPTIONS | | PRINT OPTIONS | | | | | | Page length __ | | Printer o Image o Deskjet | | Page width __ | | | | Top margin __ | | Page length __ | | Bottom margin __ | | Page width __ | | | | Top margin __ | | +-------+ --------+ | | Bottom margin __ | | Print to:|Printer| | File | | | | | +-------- +-------- | | Print to: o Printer o File| | | | | | -------+ +------+ | | -------+ +------+ | | |Cancel| | OK | | | |Cancel| | OK | | | +------+ +------- | | +------+ +------- | +------------------------------- +---------------------------+ Atari box Apple box In the four fields marked by __, you must enter the length and width of the paper you use, the top margin and the bottom margin. The top margin is the blank part above the text on a page. The bottom margin is the blank part below the text on a page. You must enter all these settings in number of characters. For instance, if you use paper 71 characters long, enter '71' at the first field. For a standard A4 paper format, you must enter '66' for the pagelength, and '80' for the page width. For standard continuous forms paper, enter '71' for the pagelength and '80' for the pagewidth. We advise for both top and bottom margin a length of 2 characters. If you use a MacIntosh, you must specify the type of your printer. If your printer is an Apple ImageWriter, you must select the [Image] button. If your printer is a HP Deskjet+, you must select the [Deskjet] button. HP Deskjet+ users should also set DIP-switch number 5 from the rightmost DIP-switch block to on. This DIP-switch block is positioned at the front of the printer. Both Atari and Apple users can specify whether they want to print to a printer or to a file. Printing to a printer means that STAR outputs directly to a printer. In this case a printer must be attached to your computer and it must be switched on and on-line. If you don't have a printer attached, but a printer attached to - 127 - Reference Guide STAR Page 8-29 8 The [ENERGYRULES] menu 29 June 1990 another computer is available, you can print to file. In this case STAR asks you to enter a filename using a file-selector. If you are not familiar with the use of a file-selector, please consult the manual that was delivered with your computer. Now STAR will make a printerfile for you. ________ ______ Possible errors: If you specify you want to print to a printer while there is no printer attached to your computer, you can not use STAR for about 30 seconds. - 128 - APPENDIX A THE PROCEDURE OF STAR In this appendix we graphically show the order in which you use the different options. The toppart of the boxes represent actions you should take at a certain moment. We numbered them 1-6. You can perform most of these actions in various ways, which we numbered 1a,1b and so on. The bottompart of the boxes represent the input for a certain action. For example to let STAR calculate a structure (action 4c), the energy rules are input. We show the options that correspond with a certain action between []. Note that we did not draw the 'returning' arrows. For example we drew no arrow from '4 GET A STRUCTURE' to '1 GET A SEQUENCE'. ___ This does not mean that you can not get a new sequence after getting a structure. However it does mean that you must have acquired a sequence before you can get a structure. - 129 - Reference Guide STAR Page A-2 The procedure of STAR 29 June 1990 +--------------------------------------+ | 1 GET A SEQUENCE | +-------------------+------------------+ | 1a Type one in |1b Open a | | yourself | sequence file | | [Edit] | [Open] | +-------------------+------------------+ | | +---------+ |sequence | +---------+ | +-----------------+-----------------+ V | V +----------------------------+ | -----------------------------+ | 2 SAVE FOR LATER USE | | | 3 SHOW OUTPUT | +-------------+--------------| | |--------------+-------------+ | 2a [Save as]| 2b [Save] | | | 3a To screen |3b To printer| | | | | | [View] | [Print] | +-------------+--------------- | +--------------+-------------+ | | +-------------+ | | energy rules| | +-------------+ V | +-------------------------------------------------|-------------+ | 4 GET A STRUCTURE | | +-------------------+---------------------+-------V-------------+ | 4a Type one in |4b Open a |4c Let STAR | | yourself | structure file | calculate one | | [Edit] | [Open] | for you | | | | [Calculate] | | | | | +-------------------+-----------+----------+--------------------+ | | +-----------+ | structure | +-----------+ | +-----------------+-----------------+ V V +----------------------------+ -----------------------------+ | 5 SAVE FOR LATER USE | | 6 SHOW OUTPUT | +-------------+--------------| |--------------+-------------+ | 5a [Save as]| 5b [Save] | | 6a To screen |6b To printer| | | | | [View] | [Print] | +-------------+--------------- +--------------+-------------+ - 130 - APPENDIX B ERRORMESSAGES In this appendix we alphabetically list all errormessages. For each message we give an explanation and what you should do if you meet that particular message. If you meet another errormessage, please write a note to us. ___ ___ ____ ____ __________ _________ ___ ________ 1. CAN NOT OPEN FILE 'filename' (FILENAME NOT ALLOWED) You tried to open a structure file from which the name is not allowed. Structure files must have the same name as the sequence you opened, except the extension must be '2' instead of '1'. _________ __ __ ________ ___ _____ 2. CHARACTER TO BE REPLACED NOT FOUND You tried to replace a nucleotide in the sequence which doesn't exist in the sequence. When you press [OK], STAR replaces nothing and you return to the main editor menu. ____ ____ 3. DISK FULL The disk you are trying to save a file to is full. Save to another disk. ____ __ __________ _______________ 4. DISK IS PHYSICALLY WRITE-PROTECTED The disk you are trying to save a file to is write protected. Remove the write protection or use another disk. ______ ______ __ ________ ______ __ ______ __ ____ 5. ERROR. DOUBLE OR NEGATIVE VALUES IN COLUMN 1. ROWS ____ __ _________ WILL BE DISCARDED You have left the editor using the [Exit] option. During the editing of bulge loop, hairpin loop, interior loop, shallow groove, deep groove or backbone energy rules you have made rows with equal values in the first column. STAR will discard the rows with equal values in the first row. ______ ______ __ ________ ______ __ ___ __ _______ 6. ERROR. DOUBLE OR NEGATIVE VALUES IN ROW 1. COLUMNS ____ __ _________ WILL BE DISCARDED You have left the editor using the [Exit] option. During the editing of shallow groove, deep groove or backbone energy rules you have made columns with equal - 131 - Reference Guide STAR Page B-2 Errormessages 29 June 1990 values in the first row. STAR will discard the columns with equal values in the first row. ______ ___ _____ __ ___ ________ __ ____ __ _________ 7. ERROR. ROW xxxxx IS NOT ALLOWED. IT WILL BE DISCARDED You have left the editor using the [Exit] option. During the editing of a structure or energy rules you have made a row that is not allowed because the row doesn't contain enough values. STAR will discard that particular row. ______ ____ ___ _______ ________ ____ _____ ______ __ 8. ERROR. STEM NOT ALLOWED (OVERLAP WITH OTHER STEM). IT ____ __ _________ WILL BE DISCARDED You have left the editor using the [Exit] option. During the editing of a structure you made a stem which overlaps with another stem. STAR will discard that stem. ______ _____ ___ _______ ________ ______ ____________ 9. ERROR. STEMS NOT ALLOWED (UNEQUAL LENGTH STEMHALVES). ____ ____ __ _________ THEY WILL BE DISCARDED You have left the editor using the [Exit] option. During the editing of a structure you made a stem with two stemhalves that are unequal in length. STAR will discard that stem. ________ __________ __ ___ ________ _________ ___ _____ 10. FILENAME 'filename' IS NOT ALLOWED. STRUCTURE NOT SAVED You used the option [Save as...] to save a structure. The filename you entered is not allowed. The first part of the filename must be equal to the name of the ________ ____ sequence file you opened. With the first part of a filename we mean the part of the name preceding the extension. The extension of the filename must begin with '2'. The extension of a filename is part of the name following the point '.'. For example: if you have opened the sequence file 'TMV.1' you can save your structure only to filenames beginning with 'TMV.2'. ________ ___ ___ __ ___ __ ______ 11. SEQUENCE TOO BIG TO FIT IN EDITOR The sequence you want to edit is too big to fit in the editor. You must enter a range you want to edit in the box that carries this message before you can start editing. ___ ______ ________ _____ ___ ___ __ ___ _____ _____ 12. THE EDITED STACKING RULES ARE NOT OF THE RIGHT SIZE. ____ ____ __ _________ THEY WILL BE DISCARDED During the editing of stacking energy rules, you changed the size of the table with stacking (it is no longer 16x16). STAR will not incorporate the new edited stacking energy rules; instead, STAR will use the stacking energy rules that were in the computer before you started the editing. - 132 - Reference Guide STAR Page B-3 Errormessages 29 June 1990 ___ ____________ ___ ____ __ ____ ___ ___ ____ __ ___ __ 13. THE ENERGY-RULES YOU WANT TO EDIT ARE TOO WIDE TO FIT IN ___ _______ ____ ____ __ _____ _________ THE EDITOR. THEY WILL BE SHOWN PARTIALLY You tried to edit shallow groove, deep groove or backbone energy rules which have too many (>10) columns to fit in the editor. STAR will show only the first 10 columns during the editing. The other columns will not be shown however they are not lost. ____ ____ __ ___ __ ________________ 14. THIS FILE IS NOT AN ENERGYRULES-FILE You tried to open a non-energy-rules file as an energy-rules file. After pressing [OK] you return to the main menu. Now you can try to open another energy-rules file. ____ ____ __ ___ _ _________ ____ 15. THIS FILE IS NOT A STRUCTURE FILE You tried to open a non-structure file as a structure file. After pressing [OK] you return to the main menu. Now you can try to open another structure file. __ ____ 16. WS FULL The computer you use has not enough memory for the operation. You must restart STAR. - 133 - APPENDIX C GLOSSARY In this appendix you find some alphabetically ordered help about technical computer and biochemistry words. ______ 1. A-knot We use the name 'a-knot' for a type of indirect stacking of stems. This type of stacking is hypothetical: the a-knot has not been found in nature so far. You can see the a-knot as an ordinary stem-loop structure of which one free end crosses the stem and basepairs with the loop. Both a 5' and a 3' a-knot can exist. We show examples in the figure below. +------+---G-+ +---U--C--C-+ | 3' | | 5' | +UGAC-?+CGCACY +AGCGAY+-CGUG 5'AA+ACUG-|-GCGUG+ +UCGCU-|-GCACAA+ 3' | | | | +-C-G--+ +--G-U-+ 3' a-knot 5' a-knot We summarized the energy values for the free end to cross the backbone in the energy rules 'Backbone'. _________ 2. APL.68000 The programming language in which we wrote STAR. _____ 3. ASCII American Standard Code for Information Interchange: a certain code that many computers use so they can communicate with each other. STAR can open sequence files originating from other programs or databases, if they are in ASCII. ________ 4. Backbone 'Backbone' is the name of the table with energy values for a connecting loop present in a-knots. - 134 - Reference Guide STAR Page C-2 Glossary 29 June 1990 _____ 5. Bulge 'Bulge' is the name for a single stranded region resulting from indirect stacking of 2 base pairs. Both 3' and 5' bulges can exist. You can see an example of a 5' bulge in the figure below. GCU A U C:G C:G +G-A:U +A-A:U C:G C:G 5'AAAY @AA+3' The names of the energy rules that STAR uses to predict a bulge are 'Stacking', 'Bulge loop' and 'Hairpin loop'. _____ ____ 6. Bulge loop 'Bulge loop' is the name of the table with energy values for bulge loops. ____ ______ 7. Deep groove 'Deep groove' is the name of the table for energy values of the connecting loop crossing the deep groove in a pseudo knot. ______ ________ 8. Direct stacking Two base pairs can stack directly or indirectly on each other. The base pairs stack directly if the stacking nucleotides are next to each other in the RNA sequence. See the figure below. | | A:U | | C:G | | ____ 9. Font A complete set of letters with a certain form. As STAR uses its own font, Apple Macintosh users must put the STAR font in the systemfile of their startup disk. _______ _____ 10. Font/DA Mover A program that Apple Macintosh users can use to move fonts between files. ____ 11. Fork 'Fork' is the name for one of the types of indirect stacking of two base pairs. You can see an example of a fork in the figure below. - 135 - Reference Guide STAR Page C-3 Glossary 29 June 1990 UAG A C U:A U:A G:C 5'AA+C:G 3'AA+A:U U:A G:C C C GUG The names of the energy rules that STAR uses to predict a fork are 'Stacking' and 'hairpin loop'. _______ 12. Hairpin 'Hairpin' is the name for one of the types of loops. You can see an example of a hairpin loop in the figure below. GCU A U C:G C:G A:U A:U 5'AAA+ +AA+3' The names of the energy rules that STAR uses to predict a hairpin are 'Stacking' and 'Hairpin loop'. _______ ____ 13. Hairpin loop 'Hairpin loop' is the name of the table with energy values for hairpin loops. ________ ________ 14. Indirect stacking Two base pairs can stack directly or indirectly on each other. The base pairs stack indirectly if nucleotides are next to each other in one strand only. See the figure below. | | A:U | @AA+ | ZAA+ G:C | | In principle, 6 types of indirect stacking be discerned. Two of them, the 3' a-knot and the 5' a-knot, are not (yet) known to exist. You can see the six types in the figures shown below. - 136 - Reference Guide STAR Page C-4 Glossary 29 June 1990 +---+ +---+ +---+ | | | | | | +? ZY @? ZY @? Z+  | | | | | |  Y | ZDD? | | | | ---+  ? | | @DY | | @DY |  | | | ZD? | | ZD? | +Y @? @DDY | | | | +--+ | | | | | | +---+ 5'-Y @+3' 5'-Y @+3' Fork 5' Bulge loop 3' Bulge loop +-------+ +----------+ +----------+ | | | 3' | | 5' | 5'------- ------|+ 3' +----+@----+ +----------+ +------------+ 5'------|-----+ +-----|-----+3'  | | | | | | +------+ +------ +-----+ pseudo- or p-knot 3'a-knot 5'a-knot ________ ____ 15. Interior loop 'Interior loop' is the name of the table with the energy rules for interior loops. _____________ 16. File-selector A box which you can use to select files. For further information: see the owners manual of your computer. ______ _______ 17. Matrix printer A printer which uses a number of metal pins to write letters on paper. _______ ____ ______ 18. Minimal loop length RNA needs a minimum length to form a hairpin-loop. This minimum length is 4 nucleotids. We have defined this by setting the energy values for loop lengths smaller than and equal to 4 nucleotides to 99.9 kcal/mol. You can find the energy values for various loop lengths in the energy rules called 'hairpin loop'. You can change these values so tha the minimum loop length will be bigger (or even smaller) than 4 nucleotids. ______ 19. P-knot 'P-knot' is the name we used for one of the ways in which two stems can stack indirectly on each other (see also 'indirect stacking'). You can see an example of a p-knot in the figure below. - 137 - Reference Guide STAR Page C-5 Glossary 29 June 1990 +-+-A-U-C-+ | | 5'--UAUCGY Z+AGGCUA|-+ 3' +AUAGC----UCCGAU+  3 | +U-C-G-C+ You can see it as an ordinary stem-loop structure of which the free 3' end crosses the shallow groove of the stem and basepairs with the loop, or the free 5' end crosses the deep groove of the stem and basepairs with the loop. We summarized the energy values used to calculate the energy it cost for the free end to cross either the deep groove or the shallow groove of a stem in the energy rules called 'deep groove' and 'shallow groove' respectively. _________ _________ 20. Secondary structure The 'secondary structure' of RNA is the assembly of structural elements which are formed by intramolecular basepairing. Besides the classical elements like stems, hairpin loops, buldge loops, interior loopsand multipleloops, STAR is able to predict some types of tertiary interactions (see 'tertiary structure'). _______ ______ 21. Shallow groove 'Shallow groove' is the name of the energy rules for one sort of p-knots. In a p-knot one loop crosses the deep groove, the other crosses the shallow groove. Following the RNA strand in a p-knot from the 3' side, the first loop you met crosses the shallow groove. Following the RNA strand in a p-knot from the 5' side, the first loop you met crosses the deep groove. We illustrate this in the figure shown below. +--------+ 5' | | | 3'>------- -------|-+ 5' 3'-----------------+ 5' -------------+ 5' >-----+ +------|--+ 3'  3 3 | 3' +--------+ shallow groove deep groove ________ 22. Stacking 'Stacking' is the name of the energy rules for the stacking of basepairs upon each other. For an example of how to calculate the stacking energy of a stem see the figure below. - 138 - Reference Guide STAR Page C-6 Glossary 29 June 1990 | | C G energy of upper pair C G lower pair A U: - 2.1 A U energy of upper pair A U lower pair C G: - 2.1 C G energy of upper pair C G lower pair G C: - 3.0 G C ---------- + 3'-Y @+ 5' - 7.2 kJ/mol Note that the energy of for example the combination 'upper pair G C and lower pair U A' is the same as for the combina- tion 'upper pair C G and lower pair A U'. If you change this value of stacking energy with the menu option [Energyrules/Edit/Stacking] you can suffice with changing only one combination. STAR changes the other automatically. ________ _____ 23. Stacking table During the calculation process, STAR accumulates all possible direct stackings in a stacking table. The size of this table is the squared length of the sequence. _________ 24. Structure The structure of a RNA molecule is defined in STAR by the nucleotide numbers of the bottom and top nucleotide pair of a stem. For example the stem in the figure below is defined by '14 17 23 26'. AUG A U C G C G U A C G 5'--AGACUCGAUAGCG+ +AUCUC-- 3' ________ _________ 25. Tertiary structure The term 'tertiary structure' is reserved here for those interactions which are formed by Watson-Crick basepairing of a loop region with a complementary region outside that loop STAR is capable of dealing with such interactions. Because relatively little is known about such interactions, we assigned a standard positive value to the free energy of the single stranded regions they create upon their formation. These energy values are summarized in the tables with energy values with the names 'deep groove', 'shallow groove' and 'Backbone'. - 139 - CHAPTER 9 CONTENTS 1 ABOUT THIS MANUAL . . . . . . . . . . . . . . . . . 3 2 HARDWARE SPECIFICATIONS . . . . . . . . . . . . . . 4 3 INSTALLING STAR AT AN ATARI ST COMPUTER . . . . . . 5 4 INSTALLING STAR AT AN APPLE MACINTOSH COMPUTER . . . 6 CHAPTER 1 INTRODUCTION CHAPTER 2 ORGANIZATION OF THE TUTORIAL CHAPTER 3 EXERCISES 3.1 EXERCISE 1 . . . . . . . . . . . . . . . . . . . . 3-1 3.2 EXERCISE 2 . . . . . . . . . . . . . . . . . . . . 3-3 3.3 EXERCISE 3 . . . . . . . . . . . . . . . . . . . . 3-7 3.4 EXERCISE 4 . . . . . . . . . . . . . . . . . . . . 3-8 3.5 EXERCISE 5 . . . . . . . . . . . . . . . . . . . 3-10 3.6 EXERCISE 6 . . . . . . . . . . . . . . . . . . . 3-12 CHAPTER 1 ABOUT THIS MANUAL 1.1 THE ORGANIZATION OF THIS MANUAL . . . . . . . . . 1-1 1.2 CONVENTIONS USED IN THIS MANUAL . . . . . . . . . 1-1 1.3 NAMING OF KEYS . . . . . . . . . . . . . . . . . . 1-3 1.3.1 Atari Keys . . . . . . . . . . . . . . . . . . . 1-3 1.3.2 Apple Keys . . . . . . . . . . . . . . . . . . . 1-3 CHAPTER 2 THE THEORY UNDERLYING STAR 2.1 INTRODUCTION . . . . . . . . . . . . . . . . . . . 2-1 2.2 FOLDING OF RNA . . . . . . . . . . . . . . . . . . 2-2 2.3 TERTIARY INTERACTIONS . . . . . . . . . . . . . . 2-3 2.4 LONG DISTANCE INTERACTIONS . . . . . . . . . . . . 2-5 2.5 THE ALGORITHM . . . . . . . . . . . . . . . . . . 2-6 - 140 - CONTENTS Page 9-2 29 June 1990 CHAPTER 3 THE PROCEDURE OF STAR 3.1 STEP 1: GET YOURSELF A SEQUENCE . . . . . . . . . 3-1 3.2 STEP 2: GET YOURSELF A STRUCTURE . . . . . . . . . 3-1 CHAPTER 4 THE STAR MENU 4.1 THE [FILES] MENU . . . . . . . . . . . . . . . . . 4-2 4.2 THE [PRIMARY] MENU . . . . . . . . . . . . . . . . 4-3 4.3 THE [SECONDARY] MENU . . . . . . . . . . . . . . . 4-4 4.4 THE [ENERGYRULES] MENU . . . . . . . . . . . . . . 4-5 4.5 SUMMARIZING: THE STRUCTURE OF THE [PRIMARY], [SECONDARY] AND [ENERGYRULES] MENUS . . . . . . . 4-6 CHAPTER 5 THE [FILES] MENU 5.1 [HELP] . . . . . . . . . . . . . . . . . . . . . . 5-1 5.2 [ABOUT STAR] (*1) . . . . . . . . . . . . . . . . 5-2 5.3 [RENAME] (*1) . . . . . . . . . . . . . . . . . . 5-3 5.4 [DELETE] . . . . . . . . . . . . . . . . . . . . . 5-4 5.5 [QUIT] . . . . . . . . . . . . . . . . . . . . . . 5-5 CHAPTER 6 THE [PRIMARY] MENU 6.1 [HELP] . . . . . . . . . . . . . . . . . . . . . . 6-1 6.2 [OPEN] . . . . . . . . . . . . . . . . . . . . . . 6-2 6.3 [SAVE AS...] . . . . . . . . . . . . . . . . . . . 6-4 6.4 [SAVE] . . . . . . . . . . . . . . . . . . . . . . 6-5 6.5 [VIEW] . . . . . . . . . . . . . . . . . . . . . . 6-6 6.6 [EDIT] . . . . . . . . . . . . . . . . . . . . . . 6-7 6.6.1 Entering The Editor . . . . . . . . . . . . . . 6-7 6.6.2 The Editor Screen And Cursor . . . . . . . . . . 6-8 6.6.3 The Editor Keys . . . . . . . . . . . . . . . . 6-8 6.6.3.1 Moving Through The Sequence . . . . . . . . . . 6-8 6.6.3.2 Deleting A Nucleotide . . . . . . . . . . . . . 6-8 6.6.3.3 Adding Nucleotides . . . . . . . . . . . . . . . 6-8 6.6.3.4 Toggle Between 'insert' And 'overwrite' Mode . 6-10 6.6.4 The Editor Menu . . . . . . . . . . . . . . . 6-11 6.6.4.1 [File] . . . . . . . . . . . . . . . . . . . . 6-11 6.6.4.1.1 [Replace] . . . . . . . . . . . . . . . . . . 6-11 6.6.4.1.2 [Glue] . . . . . . . . . . . . . . . . . . . . 6-13 6.6.4.1.3 [Exit] . . . . . . . . . . . . . . . . . . . . 6-13 6.6.4.1.4 [Quit] . . . . . . . . . . . . . . . . . . . . 6-13 6.6.4.2 The [Clipboard] Menu . . . . . . . . . . . . . 6-15 6.6.4.2.1 [Cut] . . . . . . . . . . . . . . . . . . . . 6-15 6.6.4.2.2 [Copy] . . . . . . . . . . . . . . . . . . . . 6-15 6.6.4.2.3 [Paste] . . . . . . . . . . . . . . . . . . . 6-16 6.6.4.2.4 [Unblock] . . . . . . . . . . . . . . . . . . 6-16 6.7 [PRINT] . . . . . . . . . . . . . . . . . . . . 6-17 - 141 - CONTENTS Page 9-3 29 June 1990 CHAPTER 7 THE [SECONDARY] MENU 7.1 [HELP] . . . . . . . . . . . . . . . . . . . . . . 7-2 7.2 [OPEN] . . . . . . . . . . . . . . . . . . . . . . 7-3 7.3 [SAVE AS...] . . . . . . . . . . . . . . . . . . . 7-5 7.4 [SAVE] . . . . . . . . . . . . . . . . . . . . . . 7-7 7.5 [FACTORS] . . . . . . . . . . . . . . . . . . . . 7-8 7.5.1 [Factors]: Sequential Folding Factor . . . . . . 7-8 7.5.2 [Factors]: Temperature Folding Factor . . . . . 7-9 7.5.3 [Factors]: Number Of Stems . . . . . . . . . . 7-10 7.6 [CALCULATE] . . . . . . . . . . . . . . . . . . 7-12 7.6.1 Before You Start The Calculation . . . . . . . 7-12 7.6.2 Starting The Calculation . . . . . . . . . . . 7-13 7.6.3 Generation Of All Possible Stems . . . . . . . 7-13 7.6.4 The Processing Of The Generated Stems . . . . 7-14 7.6.5 The Calculated Structure . . . . . . . . . . . 7-14 7.7 [VIEW] . . . . . . . . . . . . . . . . . . . . . 7-15 7.7.1 View Structure As Table . . . . . . . . . . . 7-15 7.7.2 View Structure By Spannings . . . . . . . . . 7-16 7.7.3 View Structure As Mountain . . . . . . . . . . 7-17 7.7.4 View Structure As Lego . . . . . . . . . . . . 7-18 7.8 [EDIT] . . . . . . . . . . . . . . . . . . . . . 7-20 7.8.1 Entering The Editor . . . . . . . . . . . . . 7-20 7.8.2 The Editor Screen And Cursor . . . . . . . . . 7-22 7.8.3 The Editor Keys . . . . . . . . . . . . . . . 7-23 7.8.3.1 Moving Through The Structure . . . . . . . . . 7-23 7.8.3.2 Deleting A Number . . . . . . . . . . . . . . 7-23 7.8.3.3 Adding A Number . . . . . . . . . . . . . . . 7-23 7.8.3.4 Toggle Between 'insert' And 'overwrite' Mode . 7-24 7.8.4 The Editor Menu . . . . . . . . . . . . . . . 7-25 7.8.4.1 The [File] Menu . . . . . . . . . . . . . . . 7-25 7.8.4.1.1 The [Replace] Option . . . . . . . . . . . . . 7-25 7.8.4.1.2 The [Glue] Option . . . . . . . . . . . . . . 7-25 7.8.4.1.3 The [Exit] Option . . . . . . . . . . . . . . 7-27 7.8.4.1.4 The [Quit] Option . . . . . . . . . . . . . . 7-28 7.8.4.2 The [Clipboard] Menu . . . . . . . . . . . . . 7-29 7.8.4.2.1 The [Cut] Option . . . . . . . . . . . . . . . 7-29 7.8.4.2.2 The [Copy] Option . . . . . . . . . . . . . . 7-30 7.8.4.2.3 The [Paste] Option . . . . . . . . . . . . . . 7-30 7.8.4.2.4 The [Unblock] Option . . . . . . . . . . . . . 7-30 7.9 [PRINT] . . . . . . . . . . . . . . . . . . . . 7-31 CHAPTER 8 THE [ENERGYRULES] MENU 8.1 [HELP] . . . . . . . . . . . . . . . . . . . . . . 8-2 8.2 [OPEN] . . . . . . . . . . . . . . . . . . . . . . 8-3 8.3 [SAVE AS...] . . . . . . . . . . . . . . . . . . . 8-5 8.4 [SAVE] . . . . . . . . . . . . . . . . . . . . . . 8-6 8.5 [VIEW] . . . . . . . . . . . . . . . . . . . . . . 8-7 8.5.1 [View]: Stacking Energy Rules . . . . . . . . . 8-9 8.5.2 [View]: Interior Loop . . . . . . . . . . . . 8-10 8.5.3 [View]: Bulge Loop . . . . . . . . . . . . . . 8-10 8.5.4 [View]: Hairpin Loop . . . . . . . . . . . . . 8-11 8.5.5 [View]: Deep Groove . . . . . . . . . . . . . 8-11 - 142 - CONTENTS Page 9-4 29 June 1990 8.5.6 [View]: Shallow Groove . . . . . . . . . . . . 8-12 8.5.7 [View]: Backbone . . . . . . . . . . . . . . . 8-12 8.6 [EDIT] . . . . . . . . . . . . . . . . . . . . . 8-13 8.6.1 Entering The Editor . . . . . . . . . . . . . 8-13 8.6.2 The Editor Screen And Cursor . . . . . . . . . 8-15 8.6.2.1 The Editor Screen While Editing Stacking Energy Rules . . . . . . . . . . . . . . . . . . . . 8-15 8.6.2.2 The Editorscreen While Editing Interior Loop Energy Rules . . . . . . . . . . . . . . . . . 8-16 8.6.2.3 The Editorscreen While Editing Bulge Loop Energy Rules . . . . . . . . . . . . . . . . . 8-17 8.6.2.4 The Editorscreen While Editing Hairpin Loop Energy Rules . . . . . . . . . . . . . . . . . 8-17 8.6.2.5 The Editorscreen While Editing Deep Groove Energy Rules . . . . . . . . . . . . . . . . . 8-18 8.6.2.6 The Editorscreen While Editing Shallow Groove Energy Rules . . . . . . . . . . . . . . . . . 8-18 8.6.2.7 The Editorscreen While Editing Backbone Energy Rules . . . . . . . . . . . . . . . . . . . . 8-19 8.6.3 The Editor Keys . . . . . . . . . . . . . . . 8-19 8.6.3.1 Moving Through The Energy Rules . . . . . . . 8-19 8.6.3.2 Deleting A Number . . . . . . . . . . . . . . 8-19 8.6.3.3 Adding A Number . . . . . . . . . . . . . . . 8-20 8.6.3.4 Toggle Between 'insert' And 'overwrite' Mode . 8-21 8.6.4 The Editor Menu . . . . . . . . . . . . . . . 8-22 8.6.4.1 The [File] Menu . . . . . . . . . . . . . . . 8-22 8.6.4.1.1 [Replace] . . . . . . . . . . . . . . . . . . 8-22 8.6.4.1.2 The [Glue] Option . . . . . . . . . . . . . . 8-22 8.6.4.1.3 The [Exit] Option . . . . . . . . . . . . . . 8-23 8.6.4.1.4 The [Quit] Option . . . . . . . . . . . . . . 8-25 8.6.4.2 The [Clipboard] Menu . . . . . . . . . . . . . 8-26 8.6.4.2.1 The [Cut] Option . . . . . . . . . . . . . . . 8-26 8.6.4.2.2 The [Copy] Option . . . . . . . . . . . . . . 8-27 8.6.4.2.3 The [Paste] Option . . . . . . . . . . . . . . 8-27 8.6.4.2.4 The [Unblock] Option . . . . . . . . . . . . . 8-27 8.7 [PRINT] . . . . . . . . . . . . . . . . . . . . 8-28 APPENDIX A THE PROCEDURE OF STAR APPENDIX B ERRORMESSAGES APPENDIX C GLOSSARY CHAPTER 9 CONTENTS - 143 - Page 9-5 29 June 1990 Sometimes your name and addres differ from that in the orderform. For example because you are the final user, but the financial department ordered the program, or because you moved in the mean time. If your adress is different, please fill out this registration card and return it to us. This will enable us to notify you of updates of STAR. Name:________________________ Phone:__________ Company:______________________________________ Adress:_______________________________________ City:________________ Country:________________ STAR licence # ______ for Atari[ ], PC[ , Mac[ ], ST ----------------------------- fold here ----------------------- Don't hesitate to write us for making suggestions, reporting problems or for comments. Comment: ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ - 144 - Page 9-6 29 June 1990 +-------+ | | | Stamp | | | | here | | | +-------+ F.H.D. van Batenburg Institute of Theoretical Biology University Leiden Kaiserstraat 63 2311 GA Leiden the Netherlands ----------------------------- fold here ----------------------- ******************************************************************** Deze diskette is samengesteld door de Stichting ST, Postbus 11129, 2301 EC Leiden. Onze bibliotheek van public domain programma's omvat op dit moment (zomer 1990) al zo'n vier honderdvijftig disks. 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