# Molecular principles of stringent response activation in bacteria

> **NIH NIH R56** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2021 · $534,248

## Abstract

PROJECT SUMMARY/ABSTRACT
As the leading cause of childhood mortality and a major cause of adult mortality, bacterial infections remain the
main threat to health worldwide. The emergence of antibiotic resistance is outpacing the development of new
antibiotics and posing a serious challenge to infectious disease treatment. Understanding how bacteria adapt to
stress during infection will spur the search for novel therapeutic approaches. To become therapeutic targets,
these mechanisms have to play key functional roles in pathogens but not in mammalian organisms.
 Bacterial “stringent response”—the master regulator of bacterial stress adaptation—drives bacterial
pathogenesis and antibiotic resistance. Stringent response depends on RelA-like proteins that make and
regulate the second messenger molecules pppGpp and ppGpp, which reformat cellular transcription to adapt to
stresses. RelA-like proteins, such as RelA and SpoT exist in most pathogenic bacteria. Despite many cellular
and biochemical studies, the mechanisms of activation of RelA-like proteins and stringent response remain
poorly understood.
 How RelA-like proteins sense stress and catalyze pppGpp synthesis and hydrolysis is unclear. RelA is
generally thought to be activated by deacyl-tRNAs, which accumulate in response to amino acid deprivation and
bind specific mRNA codons. Paradoxically, many other types of stress activate stringent response, and our
preliminary biochemical and structural studies suggest that RelA and SpoT can be activated by ribosomes in the
absence of deacyl-tRNA. The proposed studies will test the paradigm-shifting hypothesis that RelA-like proteins
sense many stresses via ribosome stalling, and that ribosomes and other activators induce distinct
conformational changes in RelA and SpoT to regulate their activities.
 This project will take advantage of recent methodological innovations and advances in biochemistry and
cryogenic electron microscopy (cryo-EM) to dissect the molecular mechanisms of two RelA-like proteins. Aim 1
will dissect the biochemical basis of the stringent response activation by RelA by identifying the functions of
regulatory domains and by kinetic characterization of RelA autoinhibition and activation by stalled ribosomes.
Aim 2 will elucidate the structural mechanisms of RelA activation by stressed ribosomes primarily using cryo-
EM. Aim 3 will dissect mechanisms of the dual enzyme SpoT, which possesses the hydrolase and weak
synthetase activities and interacts with many molecular partners via unknown mechanisms. How the ribosome
and other partners control opposing SpoT activities will be determined using biochemical and structural
approaches. Completion of this project will expand the understanding of stringent response in bacteria, and may
inform the development of new drugs to treat severe bacterial infections.

## Key facts

- **NIH application ID:** 10453921
- **Project number:** 1R56AI151372-01A1
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Andrei Korostelev
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $534,248
- **Award type:** 1
- **Project period:** 2021-08-05 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10453921

## Citation

> US National Institutes of Health, RePORTER application 10453921, Molecular principles of stringent response activation in bacteria (1R56AI151372-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10453921. Licensed CC0.

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