# Post-transcription Initiation Control of Gene Expression

> **NIH NIH R35** · PENNSYLVANIA STATE UNIVERSITY, THE · 2024 · $239,962

## Abstract

Project Summary/Abstract
 A unifying theme of my research program is to determine how RNA structure regulates gene expression
co-transcriptionally by affecting the activity of RNA polymerase (RNAP) or by affecting ribosome access to the
mRNA. Critical mechanistic insight into these processes will be obtained through comparative studies in
Escherichia coli and Bacillus subtilis by leveraging the differences of these two systems.
 RNAP pausing and transcription termination are fundamental events that regulate gene expression in all
organisms. NusA and NusG are general transcription elongation factors that have remarkable flexibility in how
they respond to pause and termination signals in bacteria, and we will determine how these two proteins
collaborate to maintain proper expression of the genome. Using RNET-seq, we identified 1600 NusG-dependent
pause sites throughout the B. subtilis genome. ~25% of these pause sites are in 5'UTRs, and the role that several
of these pauses have in regulating gene expression will be examined. The other 75% of the pause sites are in
open reading frames and the possibility that these pauses are involved in maintaining coupling of transcription
and translation will be tested. In addition, the structural basis for NusG-dependent pausing, and the cooperativity
of NusA and NusG in this process, will be investigated using cryo-EM. Whereas the KOW domain of E. coli NusG
interacts with ribosomal protein S10 for coupling of transcription and translation, this type of coupling does not
take place in B. subtilis, and the role of the B. subtilis KOW domain is unknown. However, our preliminary results
implicate this domain in co-transcriptional tRNA processing, and this exciting possibility will be examined.
Although E. coli NusG functions as an anti-pausing factor, the transcriptome-wide role of NusA on pausing has
not been examined and we will do so. Intrinsic transcription terminators consist of an RNA hairpin followed by a
U-rich tract. Using Term-seq we showed that NusA, NusG and Rho function as intrinsic termination factors in B.
subtilis. However, the roles that these proteins play in intrinsic termination in E. coli are unknown, and we will
determine those functions using engineered depletion strains for each protein.
 CsrA is a conserved RNA binding protein that binds to and regulates hundreds of mRNAs by affecting
their translation and decay. Our work indicates that it regulates numerous cellular processes, suggesting that it
is a master regulator of multiple stress responses. The extent of this regulon and how it affects metabolic changes
will be explored using a combination of in vivo CsrA-RNA footprinting, metabolomics, and regulatory studies. Of
particular importance, the footprinting methods will be applicable to essentially any organism or cell type.
 Another discovery-based theme of my research program will combine Structure-seq, a chemical probing
method for identifying RNA structural changes in vivo, with Term...

## Key facts

- **NIH application ID:** 10839515
- **Project number:** 1R35GM153190-01
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** PAUL L BABITZKE
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $239,962
- **Award type:** 1
- **Project period:** 2024-09-01 → 2029-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10839515

## Citation

> US National Institutes of Health, RePORTER application 10839515, Post-transcription Initiation Control of Gene Expression (1R35GM153190-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10839515. Licensed CC0.

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