Riboswitch Regulation of Gene ExpressionRiboswitches 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 genesThis 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 RiboswitchBacterial 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.The RNA folds intramolecularly in local regions of complementarity, presumably, while transcription is proceeding.A long untranslated leader is produced first.Nascent RNARNA polymeraseDNA templateRNA polymeraseCase 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Transcription and 3421RNA polymeraseUUUUUAUGRNA polymeraseCase 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Transcription and intramolecular RNA folding continue.34213421UUUUUAUGCase 1: Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Translation is initiated.RibosomeTypically 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. Transcription and intramolecular RNA folding continue.3421Case 2: Cellular concentration of metabolite (X) is high.Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.The alternate riboswitch conformation is stable when metabolite is bound. XXXXXRNA polymeraseXNascent RNADNA templateCase 2: Cellular concentration of metabolite (X) is high.Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.The alternate riboswitch conformation is stable when metabolite is bound. XXXXXXTranscription continues.UUUUURNA polymerase3421Case 2: Cellular concentration of metabolite (X) is high.XXXXXTranscription continues.RNA polymeraseNow, RNA folding leads to formation of an intrinsic terminator.UUUUUXX34213421Case 2: Cellular concentration of metabolite (X) is high.XXXXXTranscription continues.RNA polymeraseNow, RNA folding leads to formation of an intrinsic terminator.UUUUUXThe transcript is never completed and the metabolite biosynthetic or transport protein is not produced.3421ReviewCase 1: Metabolite is limited.Case 2: Metabolite is abundant.UUUUUXUUUUUAUGORFTranscription is completed. Biosynthetic and/or transport proteins are expressed.Transcription is terminated. Proteins are downregulated.XXX X34213421Part II:The Expanding Universe of Riboswitches an expression platform for gene regulation.A metabolite-binding aptamer domain and Riboswitch FunctionsUUUUUX3421Riboswitches were defined earlier by two main criteria: Direct (protein-free) binding of metabolite to RNA Metabolite-dependent regulation of genesThese two activities are accomplished by two functionally separate domains on the RNA: an expression platform for gene regulation.A metabolite-binding aptamer domain and Riboswitch FunctionsUUUUUX3421 Direct (protein-free) binding of metabolite to RNA Metabolite-dependent regulation of genesThese 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 Riboswitch MechanismsThis movie showed the most common case for bacterial riboswitches:Ligand binding leads to transcription termination and reduced gene expression.UUUUUX5ORFXRiboswitch MechanismsRiboswitches also regulate translation and anti-termination Ligand binding leads to transcription termination and reduced gene expression.UUUUUX5ORFLigand binding sequesters the Shine-Dalgarno sequence and reduces gene expression.Ligand binding leads to antiterminator formation and increased gene expression.UUUUUX5AUGORFXXX5AUGORFAGGAGGXRiboswitch MechanismsThey also affect RNA integrity, and perhaps splicing and stability.Ligand binding leads to mRNA cleavage by a new natural ribozyme.X5Ligand binding could control splicing in eukayotes.Possibly, ligand binding to the 3 untranslated region could affect mRNA stability.AUGORFX5AUGORFAGGUACGGAAAAAX5AUGORFXRiboswitch LigandsFigure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.There are 8 confirmed riboswitches with unique metabolite ligands. Many more conserved RNA motifs are currently