Poster abstracts
Poster number 12 submitted by Katherine Berman
Cotranscriptional RNA strand invasion mediates ligand sensing in the E. coli thiB thiamine pyrophosphate riboswitch
Katherine Berman (1Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL 60208), Julius Lucks (2Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208)
Abstract:
RNA folds immediately as it is transcribed by RNA polymerase, thereby creating RNA structures faster than new nucleotides being added to an emerging RNA chain. Consequently, cotranscriptionally folded RNA structures can differ greatly from equilibrium folded structures. Riboswitches are structured RNA elements that respond to small molecules, ions, and other metabolites to regulate transcription, translation, splicing, and mRNA degradation. Riboswitches take advantage of cotranscriptional folding to regulate gene expression by altering the folding pathway depending on the presence or absence of a ligand. The thiamine pyrophosphate (TPP) riboswitch, a highly conserved riboswitch family found in all kingdoms of life, has been shown to regulate translation via changes in cotranscriptional RNA structure. In this work, we have investigated the hypothesis that the Escherichia coli thiB TPP riboswitch functions through an RNA strand invasion mechanism by which TPP binding blocks a key rearrangement event that allows ‘sequestering stem’ to form around the ribosome binding site, preventing translation. To examine this hypothesis, we have used cotranscriptional SHAPE-seq, a technique which combines RNA chemical probing, precise polymerase arrest and high-throughput sequencing, to study intermediate structures in the folding pathway of the E. coli thiB TPP riboswitch. A series of deletion mutants created to favor or disfavor an intermediate structure predicted to facilitate this mechanism demonstrated efficient strand invasion to be essential to thiB riboswitch function. This work along with recent work on the ZTP riboswitch and the signal recognition particle (SRP) RNA have demonstrated the prevalence of strand invasion in RNA cotranscriptional folding pathways.
Keywords: SHAPE-seq, Cotranscriptional RNA folding pathways, RNA structure