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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
11:19

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Published on: February 25, 2011

Molecular mechanism of drug-dependent ribosome stalling

Nora Vazquez-Laslop1, Celine Thum, Alexander S Mankin

  • 1Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland Avenue, m/c 870, Chicago, IL 60607, USA. nvazquez@uic.edu

Molecular Cell
|April 29, 2008
PubMed

Insights

Drug-dependent ribosome stalling, essential for erm erythromycin resistance gene expression, is triggered by nascent peptide interaction with the peptidyl transferase center (PTC) and A2062 in the 23S rRNA. Macrolide antibiotics facilitate this interaction within the ribosome exit tunnel.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Biochemistry

Background:

  • Inducible expression of erm genes, conferring erythromycin resistance, depends on drug-induced ribosome stalling.
  • The precise molecular mechanisms governing this essential stalling process remain incompletely understood.

Purpose of the Study:

  • To elucidate the molecular mechanisms of ribosome stalling during the translation of the ermC leader cistron.
  • To determine the roles of the nascent peptide, macrolide antibiotic, and ribosomal components in complex formation.

Main Methods:

  • Utilized a cell-free translation system for controlled experimentation.
  • Employed toe-printing mapping, selective amino acid labeling, and site-directed mutagenesis.
  • Analyzed the ermC leader cistron translation and stalled complex formation.

Main Results:

  • Identified the peptidyl transferase center (PTC) and universally conserved nucleotide A2062 of 23S rRNA as critical for stalling.
  • Demonstrated that mutations in A2062 abolish drug-dependent stalling.
  • Showcased the nascent peptide's C-terminal sequence and macrolide antibiotic positioning within the ribosome exit tunnel as key interaction points.

Conclusions:

  • Ribosome stalling is mediated by the interaction between the nascent peptide and ribosomal elements, specifically A2062 within the PTC.
  • The macrolide antibiotic acts as a crucial modulator, positioning the nascent peptide for interaction with these sensory elements.
  • This mechanism provides insight into the regulation of antibiotic resistance gene expression.

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