Establishing Ligand Mediated RNA Folding of Translational Riboswitches as Genetic Regulators using Single Molecule Microscopy.

[摘要] Riboswitches are non-coding RNA regulatory elements located primarily in the 5’ UTR of bacterial messenger RNAs. Most commonly, they regulate gene expression of the downstream gene through transcriptional attenuation or through translational initiation inhibition in response to changing concentrations of a cellular signal. Riboswitches are composed of a ligand binding aptamer domain and an expression platform which confers the genetic decision. One of the most challenging questions remaining in the riboswitch field is how a ligand binding event can confer a large-scale conformational change, and how this change can effect a genetic decision.In this study, we demonstrate that two structurally similar transcriptional (Bsu) and translational (Tte) preQ1 riboswitches adopt similar pre-folded ensembles in the absence of ligand using a combination of single molecule techniques, molecular dynamics simulations and NMR. This result is in contrast to previous studies which suggested a largely unfolded and a loose pseudoknot for the transcriptional and translational riboswitch, respectively. Gō-model simulations of the two aptamers suggest that the ligand binds late (Bsu) and early (Tte) relative to pseudoknot folding, suggesting the two riboswitches tend to fold via conformational selection and induced fit, respectively. Finally, we show through the rational design of a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers’ pre-folded states and their ligand binding affinities. Additionally, we have developed a single molecule footprinting assay to probe the accessibility of the ribosomal binding site of two translational riboswitches as a function of ligand concentration. The feasibility of the assay is demonstrated using the V. vulnificus adenine riboswitch, in which we use a fluorophore labeled nucleic acid oligo to mimic the 3’ end of the 16S rRNA. Finally, we probe the accessibility of the Shine-Dalgarno sequence of the Tte preQ1 riboswitch in the context of its full-length mRNA, which contains two overlapping open reading frames. Our results provide a novel single molecule assay which can be extended to study other biologically relevant molecules, and provide, for the first time, a molecular basis for the occlusion of the Shine-Dalgarno sequence by translational riboswitches to effect gene regulation.

[发布机构] University of Michigan

[效力级别] Riboswitch

[关键词] Single Molecule;Riboswitch;RNA;Chemistry;Science;Chemistry