# RNA thermometers and their role in regulating Bacillus subtilis gene expression

> **NIH NIH F32** · PENNSYLVANIA STATE UNIVERSITY, THE · 2021 · $52,844

## Abstract

Project Summary/Abstract
The long-term goal of this work is to understand the virulence and heat shock responses by which bacteria
survive environmental changes. Bacteria respond rapidly to changes by regulating gene expression through an
active and complex transcriptome. Bacterial genes are often regulated by temperature-induced changes in
RNA structure, which are termed `RNA thermometers' (RNATs). RNATs often function by forming a structure
that sequesters the Shine-Dalgarno (SD) sequence, thereby preventing ribosome binding. Discovery and
verification of RNATs has historically been conducted by computational methods and in vitro studies. A gap in
knowledge exists for understanding RNAT folding in vivo, and the dynamic ability of these structures to control
gene expression. To fill this gap, global genome-wide in vivo structure probing will be applied using a method
recently developed in this laboratory called Structure-seq2. This technique will be used to probe the
transcriptome of Bacillus subtilis, a model Gram-positive bacterial species. While pioneering work led to the
description of the first RNATs, using in vivo genome-wide techniques the proposed work will identify RNATs
throughout the transcriptome, not just those near SD sequences. Using Structure-seq2 the following Aims will
be accomlished: (1) Probe the structural landscape of the B. subtilis transcriptome at low and high
temperatures to discover RNA thermometers. RNA structure is known to change in response to
temperature. This phenomenon will be explored in the B. subtilis transcriptome. As B. subtilis is a soil
bacterium, high and low temperatures will be used to replicate the extremes of natural growth in the
environment. Testing will begin at low and high temperatures of 23°C and 44°C to identify RNA structures that
change under these different conditions. This work is supported by preliminary data. (2) Classify diverse
bacterial RNA thermometers genome-wide at a series of temperatures. No RNA thermometers have been
experimentally characterized from B. subtilis, although many are predicted throughout the genome. By
conducting Structure-seq2 at a series of growth temperatures, RNATs will be discovered that have different
melting temperatures, function at narrow and wide temperature ranges, and have an instantaneous or gradual
response. Results from Aim 1 will be used as a guide for regions likely to contain RNATs. (3) Characterize B.
subtilis RNAT function. RNA structures that change in response to changing temperature will be tested as
thermometers using bgaB reporter assays. Regions of interest will be cloned as bgaB translational fusions to
report on gene expression, and transcriptional fusions will be used for in vitro transcription attenuation assays.
This work will result in the first classification of RNATs by temperature midpoints and sensitivity, and will reveal
novel regulatory strategies that will be identified in other bacterial species, including human pathogens.

## Key facts

- **NIH application ID:** 10311971
- **Project number:** 5F32GM128311-03
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** Elizabeth Jolley
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $52,844
- **Award type:** 5
- **Project period:** 2018-05-01 → 2021-09-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10311971

## Citation

> US National Institutes of Health, RePORTER application 10311971, RNA thermometers and their role in regulating Bacillus subtilis gene expression (5F32GM128311-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10311971. Licensed CC0.

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