Riboswitch Regulation of Gene Expression

1. Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004 http://www.yale.edu/breaker For citations and additional information

2. DG = 0 For a complete cycle Binding Equilibria A Leitmotif: a thermodynamic cycle couples binding to conformational change Conformational Equilibria D(DGint)

4. Riboswitches are defined by two main criteria: • Direct (protein-free) binding of metabolite to RNA • Metabolite-dependent regulation of genes Riboswitches are present in untranslated regions of mRNAs. Their purpose is to regulate gene expression in response to binding small molecule metabolites. This movie demonstrates the molecular events common to most bacterial riboswitches. Resources are listed at the end of the movie.

5. Part I: Gene Regulation by a Typical Riboswitch

6. Bacterial riboswitches are present in the 5´ untranslated region of mRNAs. Transcription is regulated by the gene promoter and transcription initiation factors …

7. RNA polymerase RNA polymerase initiates transcription. A long untranslated leader is produced first. The RNA folds intramolecularly in local regions of complementarity, presumably, while transcription is proceeding. Nascent RNA DNA template

8. 1 2 3 4 Case 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site. Transcription and … RNA polymerase RNA polymerase

9. 2 3 1 1 2 3 4 4 U U U U U A U G Case 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site. Transcription and intramolecular RNA folding continue. RNA polymerase

10. 2 3 1 4 Ribosome U U U U U A U G Case 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site. Translation is initiated. Transcription and intramolecular RNA folding continue. Typically the new mRNA codes for a biosynthetic or transport protein that raises the intracellular level of the metabolite. Gene regulation (next case) is accomplished by variations in the interactions of the regions highlighted in orange.

11. Case 2: Cellular concentration of metabolite (X) is high. RNA polymerase produces the long untranslated leader region. Intramolecular folding can lead to an alternate conformation. X X X X Nascent RNA X X RNA polymerase DNA template The alternate riboswitch conformation is stable when metabolite is bound.

12. 1 2 3 4 U U U U U Case 2: Cellular concentration of metabolite (X) is high. Transcription continues. RNA polymerase produces the long untranslated leader region. Intramolecular folding can lead to an alternate conformation. X X X X X X RNA polymerase The alternate riboswitch conformation is stable when metabolite is bound.

13. X X 1 2 1 2 3 4 3 4 U U U U U Case 2: Cellular concentration of metabolite (X) is high. Transcription continues. Now, RNA folding leads to formation of an intrinsic terminator. X X X X X RNA polymerase

14. X 1 2 3 4 U U U U U Case 2: Cellular concentration of metabolite (X) is high. Transcription continues. Now, RNA folding leads to formation of an intrinsic terminator. X X X X X RNA polymerase The transcript is never completed and the metabolite biosynthetic or transport protein is not produced.

15. Case 1: Case 2: Metabolite is limited. Metabolite is abundant. X U U U U U 1 2 3 4 ORF A U G U U U U U Review X X 2 3 1 4 X X Transcription is completed. Transcription is terminated. Proteins are downregulated. Biosynthetic and/or transport proteins are expressed.

16. Part II: The Expanding Universe of Riboswitches

17. A metabolite-binding ‘aptamer’ domain and … … an ‘expression platform’ for gene regulation. Riboswitch Functions Riboswitches were defined earlier by two main criteria: • Direct (protein-free) binding of metabolite to RNA • Metabolite-dependent regulation of genes These two activities are accomplished by two functionally separate domains on the RNA: X 1 2 3 4 U U U U U

18. A metabolite-binding ‘aptamer’ domain and … … an ‘expression platform’ for gene regulation. Riboswitch Functions Because of the modular nature of RNA structures, different types of expression platform can be linked to the conserved aptamer domain. This leads to variations in riboswitch mechanism … X 1 2 3 4 U U U U U

19. X U U U U U ORF 5´ Riboswitch Mechanisms This movie showed the most common case for bacterial riboswitches: Ligand binding leads to transcription termination and reduced gene expression. X

20. X U U U U U ORF A U G 5´ X ORF A U G 5´ Riboswitch Mechanisms Riboswitches also regulate translation and anti-termination … Ligand binding leads to transcription termination and reduced gene expression. X X U U U U U ORF 5´ Ligand binding sequesters the Shine-Dalgarno sequence and reduces gene expression. X AGGAGG Ligand binding leads to antiterminator formation and increased gene expression. X

21. X 5´ ORF A U G X GUACGG AG ORF A U G 5´ X ORF A A A A A A U G 5´ Riboswitch Mechanisms They also affect RNA integrity, and perhaps splicing and stability. Ligand binding leads to mRNA cleavage by a new natural ribozyme. X Ligand binding could control splicing in eukayotes. Possibly, ligand binding to the 3´ untranslated region could affect mRNA stability.

22. Riboswitch Ligands There are 8 confirmed riboswitches with unique metabolite ligands. Many more conserved RNA motifs are currently under investigation. Figure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.

23. Expression of tyrosine biosynthesis: T box can sense uncharged tRNA 3’CCA Cf T. Henken Codon-Anticodon tRNATyr Shine-Delgarno

24. Riboswitch Gene Regulation Riboswitches are an important mechanism of gene regulation. For example, nearly 2% of the genes of the model organism Bacillus subtilis appear to be controlled by riboswitches.

25. Metabolite Recognition Riboswitches are an economical way to see small molecules Average metabolite Coenzyme B12 tRNA Riboswitch Relative sizes of some molecules recognized for gene regulation (metabolites and tRNAs) and some agents that recognize them (riboswitches and TRAP complex). 11-mer TRAP complex

26. What have we learned? • RNA can carry out functions similar to those of proteins • Two kinds of equilibria are coordinated. • Association-dissociation • Conformational change • Linkage leads to Regulation, amplifying function

27. The End

28. David and Lucile Packard Foundation National Institutes of Health National Science Foundation Sponsors of riboswitch research in the Breaker laboratory J. Kenneth Wickiser For technical and creative assistance All members of the Breaker laboratory For helpful comments and suggestions Special thanks to:

29. Riboswitch team: