# Mechanisms of Action On Non-Coding RNA Molecules

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 2022 · $403,170

## Abstract

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 natu...

## Key facts

- **NIH application ID:** 10474534
- **Project number:** 5R01GM063162-18
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** Joseph E Wedekind
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $403,170
- **Award type:** 5
- **Project period:** 2001-04-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10474534

## Citation

> US National Institutes of Health, RePORTER application 10474534, Mechanisms of Action On Non-Coding RNA Molecules (5R01GM063162-18). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10474534. Licensed CC0.

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