The Daily Bulletin Archive

Please join the Biology Department and the Great Lakes Center for the seminar “Systematic Evaluation of Cotranscriptional RNA Folding Mechanisms Using an RNA Dependent DNA Partitioning Assay," presented by Skylar Kelley, a graduate student at the University at Buffalo, on Monday, October 2, at 3:00 p.m. in Science and Mathematics Complex 151. Attendees are welcome to arrive at 2:30 p.m. to enjoy coffee and cookies before the presentation begins.

Abstract
During transcription, RNA begins to fold as it emerges from RNA polymerase. These nascent RNA structures can regulate gene expression. Riboswitches are structured non-coding RNAs that regulate gene expression by adopting alternate structures in response to ligand binding. In addition to their role as crucial regulators of bacterial gene expression, riboswitches are essential chemical sensors and are used in developing biomolecular sensors and chemical biology tools. Despite some success in these areas, engineering riboswitches for biotechnological applications remains a challenge due to our limited understanding of how RNA folds and functions as it is synthesized during transcription. We have developed a high-throughput method called Transcription Elongation Complex Display (TECdisplay) that systematically assesses the biochemical activity of thousands of riboswitch sequence variants by quantitatively partitioning a complex DNA sequence library based on function of the encoded RNA. We demonstrate the utility of TECdisplay by characterizing the antitermination activity of a 32,768 cbe pfl ZTP riboswitch variants. The ZTP riboswitch library was designed to test the competition between the P3 stem and a putative competitor helix that was identified bioinformatically, as well as perturbing crucial terminator base pairs and the competition between the P3 stem and terminator nucleation. We find that unimpeded nucleation of base pairs that are proximal to the terminator hairpin loop promote efficient termination and ZTP riboswitch sequences likely evolved to avoid sequence combinations that promote the formation of a GC rich competitor helix. Our study demonstrates that TECdisplay is a high-throughput cotranscriptional RNA assay that can be applied to partition complex ZTP riboswitch RNA variants based on their transcription termination function. In principle, TECdisplay can be applied to any RNA function that can be used to partition a TEC library, such as RNA protein interactions.