# Mechanisms for Stress-Induced Transcriptional Reprogramming via Anti-Adaptors

> **NIH NIH R01** · BROWN UNIVERSITY · 2020 · $341,250

## Abstract

PROJECT SUMMARY
The dissociable promoter recognition subunit RpoS (also known as σs) is the master transcriptional regulator of
the general stress response in γ-proteobacteria, and plays key roles in the virulence of many pathogens,
including human, plant and animal pathogens. Under certain conditions, such as at the transition from the
logarithmic to the stationary phase or in the presence of stress signals, RpoS redirects the core RNA
polymerase machinery to a subset of promoters to reprogram transcription. However, intracellular RpoS levels
are not steady – they are low in actively dividing cells, and substantially increased upon entering the stationary
phase or upon encountering stress. To achieve proper regulation, there is tight control over RpoS levels, with
the major point of regulation occurring at the level of RpoS proteolysis by the ATP-dependent ClpXP machine.
Our central focus is to understand the mechanisms of RpoS proteolysis by ClpXP as well as its regulation by
an emerging family of proteins collectively called anti-adaptors. In order to be degraded, RpoS is presented to
ClpXP by a unique, highly specific adaptor called RssB, which acts catalytically, without being degraded. In
turn, RssB itself is regulated by interactions with stress-specific anti-adaptors. Our work will focus on the
structure and function of three-anti-adaptors: IraD (induced by oxidative stress and DNA damage), IraM
(induced by magnesium starvation) and IraP (induced by phosphate starvation). These anti-adaptors share no
sequence homology, and only weak homology with protein of known structure, warranting a structural biology
effort aimed at deciphering the underlying mechanisms of RssB recognition. We will determine the structures
of IraD, IraM and IraP, both in isolation and bound to RssB using X-ray crystallography. This will allow us to
pinpoint, at atomic resolution, residues important for anti-adaptor/RssB interactions, and also regulation of the
anti-adaptors themselves by oligomerization. We will complement these structural studies with molecular
genetics, microscopy and functional assays for protein-protein interactions and RpoS degradation, which will
allow us to correlate in vitro behavior with in vivo observations. We will also determine structures of a RpoS-
RssB-ClpXP assembly using cutting-edge methods in electron cryo-microscopy, which will allow us to
understand the RpoS and RssB conformational dynamics at the core of this paradigmatic mode of regulated
proteolysis in bacteria. Overall, this work will not only bring fundamental, mechanistic insights, but will also
open the way to the development of novel antibacterials that could target ClpXP directly, or, adaptor/anti-
adaptor interfaces. The RpoS regulon has been reported to comprise up to 10% of the Escherichia coli
genome, and RpoS itself plays important roles in bacterial persistence, host-pathogen interactions and biofilm
formation, which underlie 80% of all infections.

## Key facts

- **NIH application ID:** 9932455
- **Project number:** 5R01GM121975-04
- **Recipient organization:** BROWN UNIVERSITY
- **Principal Investigator:** Alexandra M. Deaconescu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $341,250
- **Award type:** 5
- **Project period:** 2017-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9932455

## Citation

> US National Institutes of Health, RePORTER application 9932455, Mechanisms for Stress-Induced Transcriptional Reprogramming via Anti-Adaptors (5R01GM121975-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9932455. Licensed CC0.

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