# Discovery and Characterization of New Riboswitches

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $394,262

## Abstract

Project Summary
 Riboswitches are important and ubiquitous regulatory elements that bind small-molecule effectors and
control gene expression. Many of the known riboswitch effectors have been identified based in part on the genes
under riboswitch control. However, in some cases this flow of information has been reversed, where the
identification of a riboswitch class has provided valuable insight about the function of the genes under its
regulation.
 This proposal describes the search for the ligands for new classes of riboswitches, their structural
characterization, and the molecular basis of altered specificity. We will focus on prominent RNA motifs for
which no ligand has been identified. We will determine the crystal structures of these RNAs. We will
determine which nucleotides are necessary for altered specificity and function for several riboswitches that have
variant subclasses.
 More than 50 “orphan” riboswitch candidates do not have validated ligands. Some of these orphan
riboswitch candidates present enormous opportunities to expand our knowledge of important metabolic and
signaling processes in species from various domains of life. We will pursue the identification of the ligands
for the most prominent orphan riboswitch candidates. To increase the probability that our hypotheses
regarding the ligand identity for an orphan riboswitch is strong for the largest number of orphan
candidates, we will employ a new genetic screening/selection approach to link the function of a riboswitch to
additional genes in a surrogate microorganism.
 We will pursue crystallization of multiple riboswitches as they are identified. The structures will
establish the mechanism of ligand binding and further reveal the complexity of RNA tertiary folds. Structural
analysis of ligand binding and investigation of helical switching will allow for feedback into riboswitch
search methods, facilitating discovery of previously overlooked candidates. Furthermore, each structure
will provide insights that will inform the search for riboswitch subclasses.
 There are several examples where small changes in riboswitch sequence result in dramatic
changes to the specificity of the ligand. We will pursue studies of three riboswitch families that have
variant subclasses. We have developed a massively parallel sequencing-based approach to generate
quantitative riboswitch activity profiles for thousands of individual mutants simultaneously. This work will
establish evolutionary pathways for RNA function and the energetic landscape of substrate specificity and
promiscuity. It will also aid in discovery of new subclasses and therefore new biology.

## Key facts

- **NIH application ID:** 10580082
- **Project number:** 5R01GM136969-04
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** SCOTT A STROBEL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $394,262
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10580082

## Citation

> US National Institutes of Health, RePORTER application 10580082, Discovery and Characterization of New Riboswitches (5R01GM136969-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10580082. Licensed CC0.

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