1. Riboswitches Sharon Epstein 30/03/2006 Frontiers in Metabolome sciences Feinberg Graduate School

2. Outline Introduction Concepts Evolution Structure Mechanisms Methods Examples Applications Conclusion

3. Introduction Pubmed search for “ riboswitches ” :  Reviews: 12  Articles: 57 Pubmed search for “ micro RNA ” :  Reviews: 167  Articles: 740

4. Introduction Riboswitches were “ discovered ” in the beginning of the 21 st century The idea was known but could not be proved

5. Introduction

6. Concept Riboswitches: “ method of controlling gene expression. ( … )a sequence of RNA that, through its secondary and tertiary structure, selectively binds a specific metabolite. ” (Templeton et al, 2005)

7. Concept When a metabolite is bound the secondary and tertiary structure of the RNA changes affecting transcription and translation in prokaryotes and possibly mRNA processing in eukaryotes

8. Evolution RNA world, possible mode of regulation in the absence of proteins General conservation of metabolites so far known to be involved in binding Present and studied in prokaryotes with differences in bacterial groups Present in prokaryotes mainly on 5 ’ UTR

9. Evolution Another hypothesis: it is more recent then RNA world but is present in different bacterial groups because of lateral transfer and repetitive re- invention Focus prokaryote

10. Evolution Major difference in eukaryotes: localization Present in introns and 3 ’ UTR, not well studied and not much data available Found in Arabidopsis and rice, on different splice variants (one regulated one not)

11. Structure Riboswiches are composed of two interdependent but distinct domains:  Aptamer domain (responsible for binding of ligand)  Expression plataform (responsible for impacting gene expression)

12. Structure

13. Mechanism Three known mechanisms: Formation of intrinsic terminator stem (inhibits transcription by inducing its termination) Formation of complex hiding translation initiation site Self-cleaving mechanism

14. Formation of intrinsic terminator stem

15. Inhibition of translation initiation

16. Self-cleaving mechanism

17. Methods In line probing:

18. Methods Equilibrium dialysis – radio labeled metabolite – unequal distribution RNase H probing – DNA complementary strand – conformational change – no cleavage Fluorescence – FMN quenched in contact with riboswitch

19. Examples

20. Coenzyme B12 One of the first to be discovered Upstream of cobalamin synthesis, porphyrin and cobalt transport and glutamate fermentation One of the largest aptamers with many connecting points

22. Coenzyme B12

23. Thiamine pyrophosphate Most widespread (also found by sequence similarity in plants) Identification of the riboswitch lead to function characterization of genes involved in the pathway

25. Flavin Mononucleotide Present upstream of genes for riboflavin biosynthesis and transport pathway Binds FMN 100 folds more tightly then riboflavin (difference: one phosphate)

27. Guanine and adenine Same aptamer binds both – only one point mutation C to U (forms base- pairing with ligand) Tertiary structure is similar – sequence only 59%

29. S-adenosylmethionine S-box motif – present mainly in gram- positive bacteria Upstream of sulfur, cysteine, SAM and methionine pathways 1:1 stoichiomestry, dependent on Mg+2

31. S-adenosylmethionine

32. Lysine Descriminates between l and d lysine AEC (toxic analog) is also bound by riboswitch and resistant bacteria carry mutations Potential drug target?

34. Glucosamine-6-phosphate RNA undergoes rapid self cleavage upon binding of metabolite Mutations that affect ribozyme activity de-repress the gene

36. Glycine Two aptamer upstream of glycine cleaving proteins – each binds one glycine but increases affinity of the other aptamer – increase in sensitivity Evolutionary advantage?

37. Glycine

38. Applications Drug target (antimicrobial) Molecular engineering

39. Conclusion Evolutionary clues Non coding regions of RNA as undiscovered regulatory domains New role of bioinformatics Possibilities

40. Articles Winkler WC. (2005) Winkler WC et al. (2005) Tucker BJ et al. (2005) Templeton GW et al. (2004) Soukup JK et al. (2004) Mandal M et al. (2004) Nudler E et al. (2004) Vitreschak AG et al. (2004) Kaempfer R. (2003) Lai EC. (2003) Corbino KA et al. (2005) Altman S et al. (2005)