# The role of collisions in rescuing stalled ribosomes in bacteria

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2024 · $311,688

## Abstract

PROJECT SUMMARY
 Translating ribosomes often encounter obstacles that stop them in their tracks: the synthesis of
roughly 1 out of every 250 proteins in E. coli ends in failure. Ribosome rescue factors clear stalled
ribosomes from truncated or chemically damaged mRNAs, releasing the subunits so that they can be
used again. The goal of this proposal is to define the molecular mechanisms by which rescue factors
such as tmRNA recognize stalled ribosomes without interfering with ribosomes engaged in productive
translation. For over a decade, the consensus has been that ribosome rescue factors act on truncated
mRNAs. Biochemical and structural studies indicate that tmRNA selectively reacts with ribosomes where
the mRNA tunnel downstream of the ribosomal A site is empty. Yet other critical features of the
recognition of stalled ribosomes may have been missed. A new paradigm has emerged in eukaryotes
where stalling leads to ribosome collisions and the formation of a new interface between the small
subunits of collided ribosomes. This interface is recognized by an E3 ubiquitin ligase that triggers
downstream quality control events on the mRNA. The interactions between the small subunits of bacterial
ribosomes in crystal lattices resemble those in collided eukaryotic disomes. Moreover, theoretical models
suggest that collisions play a role in lowering protein output when stalling occurs in E. coli. At present,
however, there is no direct evidence that collisions promote ribosome rescue in bacteria. We have
obtained new data using reporter mRNAs loaded with different ribosome densities that show that
ribosome collisions are required for tmRNA to rescue ribosomes stalled in the middle of an mRNA.
Furthermore, we can purify these complexes and study their composition and structure: treating cells with
an antibiotic that stalls ribosomes generates collided disomes that are nuclease-resistant. Building on
these key findings, in Aim 1 we describe unbiased approaches to identify factors that recognize stalled
ribosomes, including mass spectrometry of nuclease-resistant disomes and genetic selections against
ribosome rescue. In Aim 2, we will use ribosome profiling to follow pausing, collisions, and rescue in vivo,
asking how these phenomena change in the absence of tmRNA and novel rescue factors. Because
collisions are difficult to detect in ensemble assays, we will develop single-molecule FRET methods to
observe collisions and their effects on the binding kinetics of ribosome rescue factors. In addition, we will
determine the structure of bacterial collided disomes (and associated factors of interest). Together, these
studies will provide a comprehensive view of the role of collisions in ribosome rescue in bacteria.

## Key facts

- **NIH application ID:** 10744231
- **Project number:** 5R01GM136960-04
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Allen Rowdon Buskirk
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $311,688
- **Award type:** 5
- **Project period:** 2021-02-15 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10744231

## Citation

> US National Institutes of Health, RePORTER application 10744231, The role of collisions in rescuing stalled ribosomes in bacteria (5R01GM136960-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10744231. Licensed CC0.

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