# Small Proteins and Epitranscriptomic Factors: Emerging Mechanisms in Bacterial Gene Regulation

> **NIH NIH R35** · RUTGERS, THE STATE UNIV OF N.J. · 2024 · $390,774

## Abstract

PROJECT SUMMARY
Bacteria use a diverse set of gene regulatory mechanisms to successfully adapt to ever-changing environments.
While some regulatory pathways for gene expression such as activation or repression by transcription factors,
and transcription/translation attenuation systems are well-characterized text-book examples, others are just
coming into prominence. My research program is focused on understanding two distinct bacterial gene regulatory
mechanisms: (i) small protein regulators, less than 50 amino acids long, and (ii) epitranscriptomic proteins that
link RNA modifications and translation to metabolism and stress response. Small proteins are directly encoded
by short open reading frames, which were missed in initial genome annotations due to the preset size cut-offs
for gene size. This group of proteins are increasingly shown to play significant roles in fundamental cellular
processes such as cell division, growth and development, modulation of transport and signaling. Despite the
advances in small protein discovery, there has been little progress in functional characterization of these new-
found proteins. To tackle the major challenge in this emerging field, Theme 1 will focus on the identification and
functional characterization of small proteins involved in bacterial stress responses. We will: (i) modify and
develop ribosome-profiling methods – Ribo-RET and Ribo-RET-PUR – to measure translation rates and identify
condition-specific small proteins, (ii) develop an in vivo site-specific photo-cross-linking and a proteomics-based
approach – SPICE-MS (Small Protein Interactions via Crosslinked Ensemble Mass Spectrometry) – tailored to
capture small protein targets. Based on my previous experience with elucidating the interactions between a small
protein MgrB and its target PhoQ sensor kinase, we will systematically characterize the functions of small
proteins and their associated targets identified here using genetic and biochemical tools. Functional and
mechanistic analyses of these small proteins will be useful in designing novel antibiotics and therapeutics. In
addition, the methods developed here will be broadly applicable to small proteins from other prokaryotes as well
as eukaryotes. In theme 2, we will focus on studying epitranscriptomic enzymes and their regulatory roles.
Specifically, we will investigate role of QueE – an enzyme involved in the biosynthesis of a ubiquitous RNA
modification called queuosine – in bacterial cell division during antimicrobial peptide stress. RNA modifications
and the related machinery are modulated in response to different cellular stressors, and little is known about how
this regulation affects cell physiology. We will investigate the mechanisms by which regulation of this tRNA
modification enzyme, QueE affects cell division, translation and metabolism during antimicrobial peptide stress
response in E. coli. Together, our work will advance the fields of small protein biology and epitranscriptom...

## Key facts

- **NIH application ID:** 10906371
- **Project number:** 5R35GM147566-03
- **Recipient organization:** RUTGERS, THE STATE UNIV OF N.J.
- **Principal Investigator:** Srujana Samhita Yadavalli
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $390,774
- **Award type:** 5
- **Project period:** 2022-09-15 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10906371

## Citation

> US National Institutes of Health, RePORTER application 10906371, Small Proteins and Epitranscriptomic Factors: Emerging Mechanisms in Bacterial Gene Regulation (5R35GM147566-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10906371. Licensed CC0.

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