# Structural Elucidation of the Novel RNA Polymerase Underlying Francisella Tularensis Virulence

> **NIH NIH R21** · DUKE UNIVERSITY · 2021 · $194,778

## Abstract

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens
known. This bacterium can be readily aerosolized and utilized as a bioweapon. The morbidity and mortality of
tularemia are significant and, given the infectious capability of Francisella, a major outbreak would readily
overwhelm the ability of even the largest U.S. medical centers. Consequently, Francisella is classified as a
category A bioweapon by the US government. Genes encoded on the Francisella pathogenicity island (FPI),
are responsible for the virulence of this bacterium. The stringent starvation protein A (SspA), the macrophage
growth locus protein A (MglA) and the pathogenicity island gene regulator (PigR) mediate activation of these
genes and are therefore essential for the virulence of Francisella species that infect humans. MglA and PigR
are unique to Francisella whereas SspA proteins are found in multiple bacteria. The Francisella SspA,
however, is unusual in that it does not homodimerize but rather functions as a heterodimer with MglA. PigR is a
putative DNA binding protein with a predicted winged-helix-turn-helix motif. How SspA-MglA and PigR mediate
FPI activation is unknown, as is the underlying molecular mechanism that these proteins use to sense
infection. The overarching goal of this proposal is the molecular dissection of these mechanisms through the
study of these virulence factors in the human pathogenic Francisella tularensis tularensis and holartica
subspecies. Early studies implicated the “alarmone”, guanosine-tetraphosphate (ppGpp), as key for Francisella
virulence. We recently showed that ppGpp binds directly to MglA-SspA and unveiled the molecular details of
this interaction by solving the MglA-SspA-ppGpp complex structure. Further, we showed that ppGpp binding to
MglA-SspA mediates high affinity binding of PigR to this heterodimer. In this revised proposal, we shall
leverage our recent discoveries to dissect all components of the Francisella virulence regulatory system,
including critically, the Francisella RNA polymerase (RNAP). Our central hypothesis is that F. tularensis
employs a conceptually novel form of virulence activation involving a unique RNAP that contains the virulence
activating complex MglA-SspA as a core constituent. This is supported by ChIP-seq studies and RNAP
purifications from Francisella cells. We shall test our central hypothesis and complete the proposed objectives
through two Specific Aims. Specific Aim 1: Elucidate the high resolution structure of (MglA-SspA)-ppGpp-PigR
and identify inhibitors of ppGpp binding to MglA-SspA. Specific Aim 2: Determine the structure of Francisella
RNAP complexes by cryo-EM. The successful completion of these studies will reveal a new paradigm in
transcription regulation and enable the rational design of novel anti-Francisella-virulence therapeutics.

## Key facts

- **NIH application ID:** 10089396
- **Project number:** 5R21AI146641-02
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** RICHARD GERALD BRENNAN
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $194,778
- **Award type:** 5
- **Project period:** 2020-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10089396

## Citation

> US National Institutes of Health, RePORTER application 10089396, Structural Elucidation of the Novel RNA Polymerase Underlying Francisella Tularensis Virulence (5R21AI146641-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10089396. Licensed CC0.

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