# The RNA nanomachines of the gene expression machinery dissected at the single molecule level

> **NIH NIH R35** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $847,816

## Abstract

TITLE:
The RNA nanomachines of gene expression dissected at the single molecule level
ABSTRACT:
Over two decades, the Walter lab has contributed to the RNA field by building a broad research portfolio focused
on dissecting the mechanisms of the nanoscale RNA machines of gene expression – ranging from small viroidal
ribozymes and bacterial riboswitches to the eukaryotic spliceosome – by single molecule fluorescence
microscopy. Leveraging this expertise, the two long-term goals of the current proposal are to: 1.) Apply our
established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating
transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new
functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological
contexts and mechanisms since unexpected discoveries – as we found – often await where individual RNA
nanomachines interact. In pursuit of these goals, we will address the overarching hypothesis that dynamic RNA
structures are a major determinant of the outcomes of gene expression, often in ways that have been overlooked
by a field that historically was rooted in genetics, where genes regularly were drawn as rectangular boxes, and
function commonly was thought of as dictated by sequence rather than structure. Such thinking is countered by,
for example, the fact that nascent RNA structure has a significant impact on transcription in the form of regulatory
riboswitches embedded near the 5' ends of bacterial mRNAs and of transcription terminator hairpins at the 3'
end. Conversely, the time-ordered, 5'-to-3' directional RNA synthesis of transcription often yields kinetically
trapped RNA folds distinct from the most thermodynamically stable structure of a refolded full-length transcript.
Encapsulating the power of our pursuit, we recently combined single-molecule, biochemical and computational
simulation approaches to show that transcriptional pausing at a site immediately downstream of a riboswitch
requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced consensus pause sequence, and
electrostatic and steric interactions with the exit channel of bacterial RNA polymerase. We posit that many more
examples of such intimate structural and kinetic coupling between RNA folding and gene expression remain to
be discovered, leading to the exquisite regulatory control and kinetic proofreading enabling all life processes. To
reveal more such couplings, we will probe the dynamics of carefully purified transcriptional and translational
riboswitch-containing, as well as spliceosomal, gene expression complexes using a tailored combination of
single molecule fluorescence resonance energy transfer (smFRET), Single Molecule Kinetic Analysis of RNA
Transient Structure (SiM-KARTS) based on super-resolved co-localization of RNA targets and fluorescent
probes, cryo-electron microscopy – augmented by a proposed dye-based sing...

## Key facts

- **NIH application ID:** 10390477
- **Project number:** 5R35GM131922-04
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** NILS G WALTER
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $847,816
- **Award type:** 5
- **Project period:** 2019-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10390477

## Citation

> US National Institutes of Health, RePORTER application 10390477, The RNA nanomachines of the gene expression machinery dissected at the single molecule level (5R35GM131922-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10390477. Licensed CC0.

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