Mechanism of Transcription and Translation Regulation by Riboswitches in Bacteria

Abstract

Riboswitches are a class of structured non-coding RNA elements that have attracted attention due to their potential as anti-bacterial and anti-microbial targets upon binding to small drug-like metabolites. They are primarily found in the 5’ untranslated region of bacterial messenger RNAs (mRNAs) and regulate gene expression by changing their three-dimensional shape in response to interacting with their cognate ligands (e.g., small molecules and cellular components). This conformational change in the riboswitch’s aptamer influences its expression platform to regulate gene expression by controlling transcription and/or translation regulation. While the mechanism of ligand binding resulting in altering the aptamer structure has been extensively studied, much less is known about how this change influences the downstream expression platform or affects transcription and translation regulation. Using a range of single-molecule and biochemical assays, my doctoral work is focused on understanding riboswitch aptamers and their influence on transcription and translation. Using these assays, I studied the ligand binding and riboswitch folding mechanism of a Mn2+ sensing riboswitch that was known to regulate transcription termination. My work revealed that the riboswitch was sensitive to ligand (Mn2+) binding at sub-millimolar concentration and formed a stable docked conformation required for transcription readthrough only when Mn2+ was bound to its ligand-binding core. My observations further demonstrated that mutation of the ligand-binding core and substituting Mn2+ ions with other divalent ions did not result in such a stable docked conformation, supporting a specific role of the Mn2+ ion for the transcription regulating riboswitch. To determine the mechanism of riboswitch folding during transcriptional events like pausing of RNA polymerase (RNAP), I next studied the Mn2+ sensing riboswitch within a paused elongation complex and observed that both Mn2+ and RNAP play essential roles in co-transcriptional folding and the determination of riboswitch conformation. My data further showed that transcription factor NusA interacting with the transcribing RNA helps in signaling the riboswitch conformation to influence the outcome of transcription. Next, I studied the ligand-dependent riboswitch folding mechanism of a preQ1-sensing riboswitch and its role in determining the outcome of translation initiation. Using single-molecule studies, I found that the riboswitch precisely controls the accessibility of the ribosome binding site for 30S binding to the mRNA, a step needed to initiate translation. My data indicated that while preQ1 binding to the riboswitch both antagonizes 30S recruitment and accelerates 30S dissociation, availability of SD sequence and its distance from the riboswitch aptamer in the mRNA provided hair-triggered precision for 30S binding. Overall, my work in this dissertation leveraged single-molecule fluorescence microscopy to provide insights into the mechanisms by which riboswitches regulate gene expression at the levels of both transcription and translation beyond their aptamer-ligand interaction.