# Structural dynamics of RNAP-promoter complex in late transcription initiation

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2020 · $531,694

## Abstract

PROJECT SUMMARY:
Bacterial transcription initiation and promoter escape are highly-regulated early steps of gene expression. A
critical initiation step occurs when the 5'-end of the nascent RNA clashes with region 3.2 of the promoter
specificity factor σ70 (σR3.2) occluding the RNA exit channel. Then, either the occluded channel is cleared to
facilitate RNA forward translocation through the RNA exit channel and RNAP to escape the promoter, or the
nascent RNA back-translocates into the NTP entry channel, leading to its abortive release. We have recently
shown that a fraction of RNAPs get stabilized in a long-lived paused backtracked intermediate during
initiation. We have also shown that even after removal of σR3.2 from the RNA exit channel by the nascent
transcript, transcription kinetics is still slower than expected for elongation. Therefore, we hypothesize an
additional promoter escape-intermediate further slows down the transition from initiation to elongation, and that
both intermediates have regulatory roles. In Aim 1.A, we will elucidate the structures of the transcription initiation
complex in these states by using multiple experimentally-derived intramolecular distances as spatial constraints
on coarse-grained simulations. In Aim 1.B, we will define the molecular determinants controlling the abundance
of these late initiation intermediates. Specifically, we will examine the sequence and order in which σ70 regions
are removed from the RNA exit channel during promoter escape for different promoters. In Aim 1.B we
hypothesize that: (1) displacement of σR3 & σR4 during promoter escape follows a two-step process; (2) the
bulge formed in the scrunched DNA template strand of the transcription bubble assists in removal of these σ
regions from the RNA exit channel by projecting into the channel. We recently discovered that an excessive
number of RNAPs stall at promoters of many genes in vivo that are essential for stress-response and
that stalling is enhanced under hyperosmotic conditions in a ∆greA/∆greB E. coli strain (unpublished).
In Aim 2 we will test whether pausing in initiation occurs in live bacteria and serves as a regulatory intermediate
for stress response. We will test this hypothesis by high-resolution (1-2 nt) chromosomal DNA mapping &
footprinting in vivo techniques. We will also develop in vivo smFRET transcription bubble size assay to test
whether pausing in initiation occurs in the bacterial cell through a mechanism similar to that studied in Aim 1.
This project will significantly advance the field of transcription for the following reasons: (1) antibiotic resistance
is a serious public health concern. Elucidating the mechanisms of bacterial gene regulation is crucial for the
development of effective antimicrobial therapy; (2) the conservation of many features of RNAP structure &
function from bacteria to humans facilitates modeling of transcription mechanisms for eukaryotic enzymes; (3)
the structure of paused-backtracked RNAP...

## Key facts

- **NIH application ID:** 9857628
- **Project number:** 5R01GM130942-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** SHIMON WEISS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $531,694
- **Award type:** 5
- **Project period:** 2019-02-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9857628

## Citation

> US National Institutes of Health, RePORTER application 9857628, Structural dynamics of RNAP-promoter complex in late transcription initiation (5R01GM130942-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9857628. Licensed CC0.

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