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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
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Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria

Kazuki Saito1, Hanna Kratzat2, Annabelle Campbell1

  • 1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, USA.

Nature
|March 10, 2022
PubMed

Insights

Bacteria use a new rescue factor, SmrB, to clear stalled ribosomes. SmrB cleaves mRNA at stalled ribosomes, enabling tmRNA to rescue upstream ribosomes, preventing protein degradation.

Area of Science:

  • Bacterial molecular biology
  • Protein synthesis and regulation
  • Ribosome function and dysfunction

Background:

  • Ribosome rescue pathways are crucial for cellular homeostasis in bacteria.
  • Mechanisms distinguishing stalled from actively translating ribosomes remain poorly understood.
  • Existing pathways target problematic mRNAs and proteins for degradation.

Purpose of the Study:

  • To identify novel factors involved in bacterial ribosome rescue.
  • To elucidate the mechanism by which bacteria differentiate stalled ribosomes.
  • To understand how ribosome collisions trigger rescue pathways.

Main Methods:

  • Genetic screening in Escherichia coli to discover new rescue factors.
  • Biochemical assays to determine endonuclease activity of SmrB.
  • Cryo-electron microscopy to visualize collided ribosomes and SmrB binding sites.

Main Results:

  • Discovery of SmrB, a novel bacterial rescue factor with endonuclease activity.
  • SmrB cleaves mRNA upstream of stalled ribosomes, facilitating tmRNA-mediated rescue.
  • Ribosome collisions were identified as the trigger for SmrB recruitment and activation.
  • Cryo-EM structures revealed conserved SmrB-binding sites on collided ribosomes.

Conclusions:

  • SmrB plays a critical role in bacterial ribosome rescue by cleaving mRNA.
  • Ribosome collisions are a key signal for activating the SmrB rescue pathway.
  • This study reveals conserved mechanisms of ribosome rescue in bacteria.

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