1. 1 Ref: Ch. 5 Mount: Bioinformatics i.Protein synthesis: ribosomal RNA transfer RNA messenger RNA ii.Catalysis e.g. ribozymes iii.Regulatory molecules 17.1 Roles of RNA Molecules

2. 2 iii.Regulatory molecules Nucleic acid interactions -complementary base pairing Protein interactions -Shape -Charge -Hydrophobicity 17.2 Interactions of RNA Molecules

3. 3 -Hydrophobicity Self-complementary regions undergo complementary base pairing - depends on the formation of loops - 5’ end of one part of the molecule aligned with 3’ end of other part 17.3 Shape of RNA Molecules 5’ 3’

4. 4 17.4 Types of Structures

5. 5

6. 6

7. 7

8. 8

9. 9

10. 10 17.5 Complex Interactions Pseudoknot

11. 11 17.5 Complex Interactions Kissing hairpins

12. 12 17.5 Complex Interactions Hairpin-bulge contact

13. 13 1.The most likely structure is the most stable structure 2.Long regions of base pairing are more stable than short regions 3.GC base pairs are more stable than AU base pairs 4.Energies are estimated from experiments with synthetic RNA molecules 17.6 Principles for Predicting RNA Secondary Structure

14. 14 Energies are estimated from experiments with synthetic RNA molecules 5.The structure does not have knots 6.Loops need to contain at least 4 nucleotides 5’ 3’

15. 15 Note: Many structures are possible Formation of some loops prevents formation of other loops The stabilising effect of paired regions is balanced against the destabilising effect of unpaired regions

16. 16 The stabilising effect of paired regions is balanced against the destabilising effect of unpaired regions Sequence is drawn around circumference of circle Arcs connect paired bases Arcs don’t cross (unless pseudoknots formed) 17.7 Circle Plot

17. 17 (unless pseudoknots formed) Squiggle plot Circle plot

18. 18 WAG (Web Angis GCG) RNA secondary structure e.g. mfold Input file:VapHGRNA Output: VAPHGRNA MFOLD Use Plotfold (from WAG) to display output Save as GIF 17.8 ANGIS Programs

19. 19

20. 20

21. 21 Self-cleaving RNA molecules Catalytic activity depends on 2 o structure 17.9 Ribozymes Lehninger Principles of Biochemistry 3 rd Edition Nelson & Cox

22. 22 glmS gene produces enzyme GlmS GlmS RNA is a ribozyme – can cleave itself Ribozyme activity is greatly increased by the product of reaction catalysed by GlmS Product of GlmS enzyme shuts off synthesis of GlmS enzyme 17.10 Regulatory RNA - glmS

23. 23 Cech (2004) Nature 428:263

24. 24 CsrA protein binds to specific mRNAs and prevents their translation CsrB RNA is an antagonist of CsrA - has 18 binding sites for CsrA protein CsrA protein binds to CsrB and is not available to bind to the specific mRNAs 17.11 Regulatory RNAs – csrA, csrB

25. 25 Romeo 1998 Mol. Microbiol. 29:1321-1330

26. 26 RNA homologues maintain secondary structure - covariation of paired bases 17.12 Evolutionary Relationships 5’ 3’ A U

27. 27 Analysis of sequences of homologues for covariation can be used to predict secondary structure

28. 28 Types of secondary structure Principles for prediction of secondary structure mfold program Circle and squiggle plots Secondary structure of regulatory RNA molecules Covariation of paired bases in evolution 17.13Summary