Structural and biophysical studies of the T-box riboswitch.

Abstract

Riboswitches are noncoding structured RNAs that bind molecules and regulate gene expression in bacteria. They are commonly found in the 5' untranslated regions of bacterial messenger RNAs. T-box riboswitches bind to specific transfer RNAs (tRNAs), sense the aminoacylation state of those tRNAs, and switch conformations to regulate amino-acid metabolism and nutritional homeostasis. T-boxes are redundantly employed in bacteria to control many important operons inside a single cell, making them attractive targets for antibiotic development. Structural and biophysical characterization of this biomolecule would aid the search for T-box riboswitch inhibitors. Although high-resolution structures of some T-boxes from the glycine and atypical classes have been solved, the structure of a full-length typical T-box, the most abundant in nature, has not been achieved to date. This project has made advancements to obtain the first full length structure of a typical T-box riboswitch in complex with its cognate tRNA substrate by single particle cryo-electron microscopy. Bioinformatics analyses were performed to identify putative T-box sequences with high structural stability. This information was used to construct medium-throughput plasmid libraries of T-box-tRNA pairs for in vitro transcription. T-box-tRNA complexes were then isolated from these transcription reactions. To identify the particular sequences that formed stable complexes under the working conditions, an EMSA based assay using fluorescently labelled DNA probes was developed. The integrity of T-box-tRNA complexes was validated by dynamic light scattering and negative stain electron microscopy. These candidate sequences are currently being probed for cryo-electron microscopy studies. Moreover, this project also aims to characterize the binding kinetics of the translational class of T-boxes to tRNA using single-molecule FRET (smFRET). How the biophysical effects of this interaction have been adapted by Actinobacteria to regulate gene expression at the translational level has been elucidated by combining smFRET studies with strategic mutagenesis.