1 / 43

BCB 444/544

BCB 444/544. Lecture 25 More RNA Structure BCB 544 Projects #25_Oct19. Required Reading ( before lecture). Mon Oct 15 - Lecture 23 Protein Tertiary Structure Prediction Chp 15 - pp 214 - 230 Wed Oct 17 & Thurs Oct 18 - Lecture 24 & Lab 8 (Terribilini)

maia
Download Presentation

BCB 444/544

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. BCB 444/544 Lecture 25 • More RNA Structure • BCB 544 Projects #25_Oct19 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  2. Required Reading (before lecture) MonOct 15- Lecture 23 Protein Tertiary Structure Prediction • Chp 15 - pp 214 - 230 Wed Oct 17 & Thurs Oct 18 - Lecture 24 & Lab 8 (Terribilini) RNA Structure/Function & RNA Structure Prediction • Chp 16 - pp 231 - 242 Fri Oct 18- Lecture 25 (& Mon Oct 22) Gene Prediction • Chp 8 - pp 97 - 112 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  3. Homework Assignment ALL: HomeWork #4(emailed & posted online Sat AM) Due: Mon Oct 22 by 5 PM (not Fri Oct 19) Read: Ginalski et al.(2005) Practical Lessons from Protein Structure Prediction, Nucleic Acids Res. 33:1874-91.http://nar.oxfordjournals.org/cgi/content/full/33/6/1874 (PDF posted on website) • Although somewhat dated, this paper provides a nice overview of protein structure prediction methods and evaluation of predicted structures. • Your assignment is to write a summary of this paper - for details see HW#4 posted online & sent by email on Sat Oct 13 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  4. BCB 544 Only: New Homework Assignment 544 Extra#2(posted online Thurs?) Due: Fri Nov 2 by 5 PM HW#2 is next step in Team Projects Will end lecture a few minutes early today - to allow time to meet & discuss 544 Teams& Projects BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  5. Seminars this Week BCB List of URLs for Seminars related to Bioinformatics: http://www.bcb.iastate.edu/seminars/index.html • Oct 18 Thur - BBMB Seminar 4:10 in 1414 MBB • Sachdeve Sidhu(Genentech)Phage peptide and antibody libraries in protein engineering and ligand selection • Was great talk! • Oct 19 Fri - BCB Faculty Seminar 2:10 in 102 ScI • Lyric Bartholomay(Ent, ISU) Computational Biology and vector-borne disease: from the field to the bench BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  6. Chp 16 - RNA Structure Prediction SECTION V STRUCTURAL BIOINFORMATICS Xiong: Chp 16RNA Structure Prediction (Terribilini) • RNA Function • Types of RNA Structures • RNA Secondary Structure Prediction Methods • Ab Initio Approach • Comparative Approach • Performance Evaluation BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  7. RNA Function This slide has been changed • Storage/transfer of genetic information • Newly discovered regulatory functions • miRNA & si RNA pathways, especially • Catalytic BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  8. RNA types & functions BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  9. RNA Structures • RNA forms complex 3D structures • Mainly "single-stranded" - but: • Single RNA strandscan self-hybridize to form Base-paired regions BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  10. Levels of RNA Structure This slide has been changed Like proteins, RNA has primary, secondary, and tertiary structure (& quaternary structure, too) • Primary structure = Ribonucleotide sequence • Secondary structure = Helix vs turn (base-paired vs single-stranded) Note: in RNA, helices often involve long-range interactions • Tertiary structure = 3D structure (also due to long-range interactions) • Quaternary structure = complex of 2 or more RNA strands Rob Knight Univ Colorado BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  11. Common structural motifs in RNA • Helices • Loops • Hairpin • Interior • Bulge • Multibranch • Pseudoknots • Tetraloops Fig 6.2 Baxevanis & Ouellette 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  12. Covalent & non-covalent bonds in RNA This is a new slide • Primary: • Covalent bonds • Secondary/Tertiary • Non-covalent bonds • H-bonds • (base-pairing) • Base stacking Fig 6.2 Baxevanis & Ouellette 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  13. RNA Structure Prediction This slide has been changed • RNA tertiary structure is very difficult to predict • Focus on predicting RNA secondary structure: • Given an RNA sequence, predict its secondary structure • Almost all methods ignore higher order secondary structures such as pseudoknots& tetraloops • Specialized software is available for predicting these BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  14. RNA Pseudoknots & Tetraloops This is a new slide • Often have important regulatory or catalyltic functions Pseudoknot Tetraloop http://www.lbl.gov/Science-Articles/Research-Review/Annual-Reports/1995/images/rna.gif http://academic.brooklyn.cuny.edu/chem/zhuang/QD/mckay_hr.gif BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  15. Base Pairing in RNA This slide has been changed See: IMB Image Library of Biological Molecules G-C, A-U, G-U ("wobble") & many variants http://www.fli-leibniz.de/ImgLibDoc/nana/IMAGE_NANA.html#basepairs BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  16. Experimental RNA structure determination? • X-ray crystallography • NMR spectroscopy • Enzymatic/chemical mapping BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  17. RNA Secondary Structure Prediction Methods This slide has been changed Two (three, recently) main types of methods: • Ab initio - based on calculating most energetically favorable secondary structure(s) Energy minimization (thermodynamics) • Comparative approach - based on comparisons of multiple evolutionarily-related RNA sequences Sequence comparison (co-variation) • Combinedcomputational & experimental Use experimental constraints when available BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  18. RNA Secondary structure prediction - 1 This is a new slide Energy minimization (thermodynamics) • Algorithms: Dynamic programming to find high probability pairs (also, some Genetic algorithms) • Software: Mfold - Zuker RNAfold (Vienna Package) -Hofacker RNAstructure - Mathews Sfold - Ding & Lawrence R Knight 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  19. RNA Secondary structure prediction - 2 This is a new slide 2) Comparative sequence analysis (co-variation) • Algorithms: Mutual information Context-free grammars • Software: RNAlifold Foldalign Dynalign BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  20. RNA Secondary structure prediction - 3 This is a new slide DMS G 200 220 240 Kethoxal modification (mild) (strong) DMS modification (mild) (strong) 3) Combined experimental & computational • Experiments: Map single-stranded vs double-stranded regions in folded RNA • How? Enzymes: S1 nuclease, T1 RNase Chemicals: kethoxal, DMS, OH • Software: Mfold Sfold RNAStructure RNAFold RNAlifold BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  21. 1 - Ab Initio Prediction This slide has been changed • Requires only a single RNA sequence • Calculates minimum free energy structure • Base-paired regions have lower free energy, so methods "attempt to find secondary structure with maximal base pairing" (Careful!) • IMPORTANT: Largest contribution to energy is to nearest neighbor (base-stacking) interactions, not base-pairing! BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  22. Ab Initio Prediction: Clarifications This slide has been changed • Free energy is calculated based on parameters determined in the wet lab • Correction: Use known energy associated with each type of nearest-neighbor pair (base-stacking) (not base-pair) • Base-pair formation is not independent: multiple base-pairs adjacent to each other are more favorable than individual base-pairs - cooperative - because of base-stacking interactions • Bulges and loops adjacent to base-pairs have a free energy penalty BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  23. Ab Initio Prediction: What are the assumptions? This is a new slide • Native tertiary structure or "fold" of an RNA molecule is (one of) its "lowest" free energy configuration(s) Gibbs free energy = G in kcal/mol at 37C = equilibrium stability of structure lower values (negative) are more favorable Is this assumption valid? in vivo? - this may not hold, but we don't really know BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  24. Energy minimization: What are the rules? This is a new slide Basepair Basepair A U A U A=U A=U G = -1.2 kcal/mole A U U A A=U U=A G = -1.6 kcal/mole What gives here? Why 1.2 vs 1.6? C Staben 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  25. Energy minimization calculations:Base-stacking is critical This is a new slide - Tinocco et al. C Staben 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  26. Ab initio RNA Structure Prediction: Uses Nearest-neighbor parameters This is a new slide • Most methods for ab initio prediction (free energy minimization) use nearest-neighbor energy parameters (derived from experiment) for predicting stability of an RNA secondary structure (in terms of G at 37C) & most available software packages use same set of parameters - Mathews, Sabina, Zuker BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  27. Ab Initio Energy Calculation This slide has been changed • Search for all possible base-pairing patterns • Calculate total energy of each structure based on all stabilizing and destabilizing forces Total free energy for a specific RNA conformation = Sum of incremental energy terms for: • helical stacking (sequence dependent) • loop initiation • unpaired stacking (favorable "increments" are < 0) Fig 6.3 Baxevanis & Ouellette 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  28. Dot Matrices • Can be used to find all possible base pair patterns • Compare input sequence to itself and put a dot where there is a complimentary base R Knight 2005 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  29. Dynamic Programming This slide has been changed • Finding optimal secondary structure is difficult - lots of possibilities • Compare RNA sequence with itself • Apply scoring scheme based on energy parameters for base stacking, cooperativity, and penalties for destabilizing forces • Find path that represents most energetically favorable secondary structure BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  30. Problem with DP Approach • DP returns SINGLE lowest energy structure • There may be many structures with similar energies • Also, predicted secondary structure is only as good as energy parameters used • Solution: return multiple structures with near optimal energies BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  31. Popular Ab Initio Prediction Programs • Mfold • Combines DP with thermodynamic calculations • Fairly accurate for short sequences, less accurate as sequence length increases • RNAfold • Returns multiple structures near predicted optimal structure • Computes larger number of potential secondary structures than Mfold, so uses a simplified energy function BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  32. 2 - Comparative Prediction Approaches • Use multiple sequence alignment • Assume related sequences fold into same secondary structure BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  33. Co-variation patterns in MSAs are critical • RNA functional motifs are conserved • To maintain RNA structure during evolution, a mutation in a base-paired residue must be compensated for by a mutation in residue with which it pairs • Comparative methods search for co-variation patterns in MSAs BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  34. Consensus Structures • Predict secondary structure of each individual sequence in a MSA • Compare all structures and try to identify a consensus structure BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  35. Popular Comparative Prediction Programs Two main types: • Require user to provide MSA • RNAalifold • No MSA required • Foldalign • Dynalign BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  36. RNAalifold • Requires user to provide MSA • Creates a scoring matrix combining minimum free energy and co-variation information • DP used to identify minimum free energy structure BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  37. Foldalign • User provides pair of unaligned RNA sequences • Constructs alignment & computes conserved structure • Suitable only for relatively short sequences BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  38. Dynalign • User provides two unaligned input sequences • Calculates possible secondary structures using algorithm similar to Mfold • Compares multiple structures from both sequences to find a common structure BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  39. 3 - Popular Programs that use Combined Computational Experimental Approaches • Mfold • Sfold • RNAStructure • RNAFold • RNAlifold BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  40. SL Y SL Y SL Z SL X SL Z SL X Sfold -51.14 kcal/mol Mfold -54.84 kcal/mol SL Y SL Z SL Y SL X SL Z SL X RNAstructure -71.3 kcal/mol RNAfold -80.16 kcal/mol Comparison of Predictions for Single RNA using Different Methods JH Lee 2007 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  41. Comparison of Mfold Predictions: -/+ Constraints Mfold -126.05 kcal/mol Mfold plus constraints -54.84 kcal/mol JH Lee 2007 BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  42. Performance Evaluation This slide has been changed • Ab initio methods? correlation coefficient = 20-60% • Comparative approaches? correlation coefficient = 20-80% • Programs that require user to supply MSA are more accurate • Comparative programs are consistently more accurate than ab initio • Base-pairs predicted by comparative sequence analysis for large & small subunit rRNAs are 97% accurate when compared with high resolution crystal structures! - Gutell, Pace • BEST APPROACH?Methods that combine computational prediction (ab initio & comparative) with experimental constraints (from chemical/enzymatic modification studies) BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

  43. BCB 544 "Team" Projects • 544 Extra HW#2 is next step in Team Projects • Write ~ 1 page outline • Schedule meeting with Michael & Drena to discuss topic • Read a few papers • Write a more detailed plan • You may work alone if you prefer • Last week of classes will be devoted to Projects • Written reports due: Mon Dec 3(no class that day) • Oral presentations (15-20') will be:Wed-Fri Dec 5,6,7 • 1 or 2 teams will present during each class period • See Guidelines for Projects posted online BCB 444/544 F07 ISU Dobbs #25 - More RNA Structure & BCB 544 Projects

More Related