Sunday, May 11, 2025
5/11/2025
Mechanisms of Action On Non-Coding RNA Molecules - Project Summary RNA plays a central role in many gene-regulatory processes and is viewed as an important drug target. Riboswitches are natural RNA sensors typically found in bacterial mRNAs where they bind cognate metabolites. This process elicits riboswitch conformational changes that control expression of downstream genes. Although most riboswitches bind only a single ligand, we discovered that the Type I preQ1-I (class I) riboswitch recognizes two metabolites, leading to positive cooperativity that extends the riboswitch’s preQ1-sensing range in bacterial cells. The discovery that a small, single-domain RNA can bind two metabolites is unprecedented in the field. The importance of this discovery is heightened by the fact that the Type I sub-class is the most prominent preQ1- sensing riboswitch in the biosphere and exists in many human pathogens. Surprisingly, cooperative binding by the Type I preQ1-I riboswitch has gone undetected for more than a decade — until now. The overarching goal of this proposal is to define the underlying molecular attributes that confer cooperativity and its interplay with preQ1- sensing during bacterial gene regulation. To address this challenge, we developed innovative tools including: (i) adaptation of a bacterial reporter assay in which the Type I preQ1 riboswitch controls GFPuv expression, which revealed two EC50 values for preQ1 binding; (ii) development of software for analysis of cooperative isothermal titration calorimetry (ITC) data, which yields microscopic binding constants whose ratio quantifies cooperativity; and (iii) refinement of our GFPuv reporter coupled in cell (ReCo-ic)SHAPE (selective 2´-hydroxyl acylation analyzed by primer extension) assay to pinpoint functionally-relevant, preQ1-dependent riboswitch conformations in live bacteria. These approaches form a rigorous foundation to define the chemical determinants of cooperative ligand binding and to evaluate their effects on biological function. We will address our central goal in three aims: (1) Determine crystal structures of Type I preQ1 riboswitches in apo and bound states; (2) Define riboswitch chemical attributes that confer cooperative binding and evaluate their role in bacterial gene regulation; and (3) Define gene-regulatory conformational changes using ReCo-icSHAPE in concert with all-atom computational approaches to delineate cooperative binding pathways. We are a team of experts with strong records in: RNA crystallography, effector binding, RNA chemical modification and bacterial reporter assays (Wedekind, P.I.); RNA dynamics and computational prediction of RNA structure using experimental restraints (Mathews, co-I); next- generation sequencing (Pritchett, collaborator); and biophysical approaches (Jenkins, collaborator). Given our novel premise, expertise and team synergy, we are uniquely qualified to perform this work. High-value outcomes include a new structural and chemical understanding of cooperative binding by the smallest natural riboswitch aptamer, which we hypothesize is a hallmark of the entire Type I sub-class. Overall, the proposed analysis will broaden our understanding of RNA-mediated gene regulation, which has implications for antibacterial targeting.
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