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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
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A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging

Brian D Janssen1, Fernando Garza-Sánchez, Christopher S Hayes

  • 1Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.

Plos One
|November 22, 2013
PubMed

Insights

The enzyme cleaving A-site mRNA in Escherichia coli is unknown, but its activity doesn't affect tmRNA-SmpB ribosome rescue. This suggests A-site cleavage may aid other mRNA recycling pathways.

Area of Science:

  • Molecular Biology
  • Bacterial Genetics
  • Protein Synthesis

Background:

  • Prolonged translational arrest in Escherichia coli leads to mRNA degradation at the A site of stalled ribosomes.
  • This A-site cleavage is linked to translation quality control, involving the tmRNA-SmpB system for ribosome rescue and ssrA-peptide tagging for protein degradation.
  • The specific enzyme responsible for A-site mRNA cleavage remains unidentified.

Purpose of the Study:

  • To investigate the impact of A-site mRNA cleavage on the efficiency of the tmRNA-SmpB ribosome rescue system.
  • To determine if A-site truncated transcripts are essential for tmRNA-SmpB-mediated ribosome rescue and ssrA-peptide tagging.

Main Methods:

  • Utilized a model transcript in Escherichia coli designed to undergo stop-codon cleavage during inefficient translation termination.
  • Quantified ssrA-peptide tagging of the encoded protein in wild-type (rnb(+)) and RNase II-deficient (Δrnb) strains.
  • Performed pulse-chase analysis to assess ribosome recycling rates from truncated transcripts in both rnb(+) and Δrnb cells.

Main Results:

  • A-site mRNA cleavage was reduced approximately three-fold in Δrnb cells compared to rnb(+) cells.
  • The efficiency of ssrA-peptide tagging was identical in both rnb(+) and Δrnb backgrounds.
  • Ribosome recycling from test transcripts occurred at similar rates in both rnb(+) and Δrnb cells.

Conclusions:

  • A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue.
  • The A-site mRNA cleavage process may be involved in alternative mRNA or ribosome recycling pathways distinct from tmRNA-SmpB.
  • Further research is needed to elucidate the precise role and mechanism of A-site mRNA cleavage in bacterial translation regulation.

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