Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004

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

Part I: Gene Regulation by a Typical Riboswitch

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

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 RNA polymerase DNA template

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

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

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

X X X X X X 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.

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

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 X X 3 4 2 1 3 4 2 1 RNA polymerase U

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

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

The Expanding Universe Part II: The Expanding Universe of Riboswitches

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

Riboswitch Functions 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: 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 … A metabolite-binding ‘aptamer’ domain and … … an ‘expression platform’ for gene regulation. X 1 2 3 4 U U U U U

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

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

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

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.

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.

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

The End

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

Riboswitch team: Ron Breaker Jeff Barrick Gail Emilsson Izabela Puskarz Ben Boese Mark Lee Adam Roth Keith Corbino Jinsoo Lim Narasimhan Sudarsan Smadar Cohen-Chalamish Maumita Mandal Ken Wickiser Margaret Ebert Shingo Nakamura Wade Winkler Ali Nahvi

For citations and additional information For more information: http://www.yale.edu/breaker For citations and additional information