Functional Roles of Nascent RNA Structure in Regulating and Coordinating Gene Expression

Gene expression relies on a complex series of cellular events, many of which can be influenced by cis-acting messenger RNA (mRNA) structures that can influence and sometimes regulate transcription, translation and mRNA degradation in prokaryotes, and splicing, polyadenylation and other RNA processing events in eukaryotes. Many of these processes, as well as the folding and remodeling of mRNA structures, can occur cotranscriptionally. This motivates our long-term goal to test the hypothesis that cotranscriptional RNA folding, and interactions with cellular factors, establishes nascent mRNA structures that functionally orchestrate gene expression by regulating and coordinating central steps of mRNA processing. Towards this goal, we are pursuing one aspect of this idea through detailed structure-function studies of transcriptional riboswitches: elements in the 5’ leaders of bacterial mRNAs that contain overlapping ligand-binding aptamer and expression platform domains that together cotranscriptionally mediate ligand-dependent transcription regulation. Because of this, riboswitches are important model systems for understanding how nascent RNA folding impacts gene expression. They are also important given their roles as a broadly distributed regulatory mechanism across domains of life, their promise as antibiotic targets, their importance for studying RNA-small molecule interactions, and their potential as components of biotechnologies. While aptamer-ligand interactions have been elucidated, big questions remain as to how dynamic RNA structural changes communicate across the entire riboswitch to mediate a regulatory decision, and how this structural communication pathway is influenced by sequence variation in the aptamer and expression platforms. This proposal details a set of specific aims that addresses these fundamental questions: (1) how does sequence covariation in aptamer-expression platform overlap tune function, (2) how do peripheral aptamer helices tune ligand binding and cotranscriptional folding events, and (3) how does expression platform variation alter riboswitch folding and function? To address these, we have developed a discovery and characterization framework to study the dynamic structure-function principles of the fluoride and preQ1 riboswitches that utilize pseudoknot-mediated ligand recognition: (i) in vivo sort-seq and bioinformatics to rapidly identify functional sequence variations, (ii) cotranscriptional SHAPE-Seq (selective 2’-hydroxyl acylation analyzed by primer extension sequencing) to characterize ligand dependent cotranscriptional folding pathways at nucleotide resolution, (iii) biophysical methods (calorimetry and stopped-flow kinetics) to measure ligand binding properties, (iv) transcription assays to uncover transcription pausing and kinetics, and (v) a framework to uncover and apply mechanistic rules to test specific sequence-structure-function principles. Our approach will establish a new framework for understanding riboswitch mechanism, and more broadly the roles of nascent RNA structures in influencing bacterial gene expression. It will also lay the groundwork to investigate these roles in more complex eukaryotic processes.