Wednesday, January 15, 2025
1/15/2025
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.
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