# Kinetic regulation of mycobacterial transcription

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $393,750

## Abstract

PROJECT SUMMARY/ABSTRACT
 Transcription in all bacteria is achieved by a single core RNA polymerase (RNAP) which associates with
a σ-subunit to form an RNAP holoenzyme, bind DNA promoter sequences, and initiate transcription. Most
transcriptional regulation occurs at the level of initiation and transcription factors mediate this regulation by
directly modulating the interaction between RNAP and the promoter, manipulating the rates of interconversion
between closed and open RNAP-promoter complexes (RPc and RPo respectively), or affecting the rate of
promoter escape. We have recently shown that transcription in Mycobacterium tuberculosis (Mtb) is considerably
different from that in the model bacterium E. coli in that Mtb RNAP forms inherently unstable RPo complexes as
compared to E. coli RNAP. Furthermore, Mtb possess two essential transcription factors, CarD and RbpA, that
are absent from E. coli. We have previously shown that these factors stabilize mycobacterial RPo, albeit through
different mechanisms, and are able to cooperatively and dramatically change the kinetics of Mtb RNAP RPo
formation such that it mirrors those of E. coli RNAP. We have been studying CarD and RbpA activities in vivo
and in vitro and have proposed kinetic mechanisms for each factor in the context of the housekeeping σA RNAP
holoenzyme on the ribosomal RNA (rRNA) rrnAP3 promoter. Our studies have been instrumental in
understanding the fundamental properties of these essential transcription factors and have revealed insight into
unique properties of Mtb transcription. However, CarD and RbpA activities have only been examined on a handful
of mycobacterial promoters with sequence elements similar to promoters found in E. coli even though in general
Mtb promoters differ considerably from those in E. coli. In addition, CarD has only been studied in the context of
the σA RNAP holoenzyme, despite the importance of the 12 Mtb alternative σ-factors that regulate the bacterias
response to stresses encounter during pathogenesis. Thus, we still do not know how CarD and RbpA affect
expression of the vast majority of genes within the Mtb chromosome and how this regulation contributes to
viability under the conditions Mtb experiences during infection. In this project, we will measure the kinetics of Mtb
initiation using rapid-mixing stopped-flow techniques as well as high-resolution single-molecule approaches to
address how CarD and RbpA differentially affect gene expression from diverse promoters and in the context of
alternative holoenzymes essential for bacterial stress responses enacted during infection. Our Aims are to: (1)
Test the hypothesis that CarD and RbpA can either activate or repress transcription depending on promoter
context, (2) Expand our kinetic model of factor-dependent mycobacterial transcription initiation, and (3)
Determine how CarD and RbpA are influenced by alternative sigma-factors. Completion of these Aims will result
in a holistic view of Mtb transcriptional ...

## Key facts

- **NIH application ID:** 9982385
- **Project number:** 5R01GM134362-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Eric A Galburt
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $393,750
- **Award type:** 5
- **Project period:** 2019-08-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9982385

## Citation

> US National Institutes of Health, RePORTER application 9982385, Kinetic regulation of mycobacterial transcription (5R01GM134362-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9982385. Licensed CC0.

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