# Structural and Functional Characterization of RNA polymerase and its Regulators from Mycobacterium tuberculosis and Clostridioides difficile

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2022 · $177,103

## Abstract

Project Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a major health problem.
The Center for Disease Control estimates that approximately 1/3 to 1/4 of the world’s population is latently
infected. RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for the
Rifamycin (Rif) class of antibiotics, a first line therapeutic treatment for TB. RNAP is thus a proven and
attractive target for the development of new drugs. This highlights the importance of our recent structural and
functional characterization of Mtb RNAP and the roles of two essential transcription factors required for full
transcriptional activity. The previous grant enabled us to provide a 2.8 Å resolution crystal structure of an
RNAP transcription initiation complex (TIC) from M. smegmatis and more recently cryo-EM structures of Mtb
transcription complexes. In this proposal, cryo-EM will be used to examine RNAP complexes as a starting point
to elucidate the mechanisms of a family of relatively uncharacterized transcription factors, the WhiB factors.
The WhiB factors are only found in Actinobacteria and have roles in Mtb that include essentiality for growth and
division, and responses to host induced stresses including antibiotic tolerance, nitric oxide, macrophage
invasion and reactive oxygen species. We will use a multidisciplinary approach that includes structural,
biochemical, genomic and in vivo experiments (in collaboration with J. Rock) to understand the roles and
mechanism of this important, but relatively uncharacterized family of transcription factors. The results from the
aims here have the potential to not only elucidate the mechanism and biology of these factors, but also provide
a platform for new targets for clade-specific antibiotic development and serve to guide us on how to increase
the efficacy of the current repertoire of antibiotics.
The results from the previous funding period have led to high resolution structures of Mycobacteria RNAP (by
cryo-EM and crystallography), and provided the opportunity to characterize how Rif and Rif derivatives that
inhibit Rif resistant (RifR) bacteria inhibit Mycobacteria RNAP. Here we propose to continue this line of research
with structurally uncharacterized Rif derivatives, provided by S. Brady, that inhibit additional RifR Mtb RNAPs.
Clostrioides difficile (Cdiff), a Gram-positive, sporulating, anaerobic bacterium, is an opportunistic pathogen
which is deadly to compromised hosts. Fidaxomicin (Fdx), the only other FDA approved antibiotic which targets
RNAP, is a powerful treatment for Cdiff infection. Our recent work established that Fdx can inhibit Mtb RNAP
potently, but that potency is dependent on the Actinobacteria-specific transcription factor RpbA, which is
absent in Cdiff. Here we propose to extend our expertise in biochemical and structural studies of bacterial
RNAPs to include the previously uncharacterized clade of Firmicutes to which Cd...

## Key facts

- **NIH application ID:** 10581925
- **Project number:** 3R01GM114450-08S1
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** ELIZABETH A CAMPBELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $177,103
- **Award type:** 3
- **Project period:** 2015-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10581925

## Citation

> US National Institutes of Health, RePORTER application 10581925, Structural and Functional Characterization of RNA polymerase and its Regulators from Mycobacterium tuberculosis and Clostridioides difficile (3R01GM114450-08S1). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10581925. Licensed CC0.

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