A newborn RNA switches its fate

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Abstract

Riboswitches enable microbes to rapidly respond to changing levels of metabolites. A high-throughput platform reveals how RNA structural transitions kinetically compete during transcription in a new mechanism for riboswitch function.

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Most cited references 8

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Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.

Kevin Wilkinson, Edward Merino, Kevin Weeks (2006)

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.

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The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.

Thomas Winkler, D Crothers, Ronald R. Breaker … (2005)

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

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Direct observation of cotranscriptional folding in an adenine riboswitch.

Kirsten L Frieda, Steven Block (2012)

Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These experiments furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.