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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

Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

Dmitry E Burakovsky1, Petr V Sergiev, Maria A Steblyanko

  • 1Department of Chemistry, Moscow State University, Moscow, 119899, Russia.

RNA (New York, N.Y.)
|July 30, 2010
PubMed

Insights

Mutations in the ribosome's 23S rRNA accommodation gate did not hinder aminoacyl-tRNA (aa-tRNA) binding or peptide bond formation. However, these changes impaired the release of peptides by release factor 2 (RF2).

Area of Science:

  • Molecular Biology
  • Ribosome Function
  • Protein Synthesis

Background:

  • Aminoacyl-tRNA (aa-tRNA) and release factors (RF1, RF2) bind the ribosome's 50S subunit for peptide bond formation or hydrolysis.
  • Computer simulations identified 23S rRNA nucleotides (U2492, C2556, C2573) forming a gate that may retard aa-tRNA accommodation.

Purpose of the Study:

  • To investigate the role of 23S rRNA nucleotides C2573 and A2572 in aa-tRNA binding, peptide bond formation, and RF2-dependent peptide release.
  • To determine the impact of mutations in the putative accommodation gate on ribosome function.

Main Methods:

  • Site-directed mutagenesis of 23S rRNA nucleotides C2573 and A2572.
  • Assays for aa-tRNA binding and accommodation.
  • Measurement of peptide bond formation.
  • Analysis of RF2-dependent peptide release.

Main Results:

  • Mutations at C2573 and A2572 did not affect aa-tRNA accommodation, peptide bond formation, or aa-tRNA selection fidelity.
  • These mutations significantly impaired RF2-catalyzed peptide release.
  • The ribosome demonstrates robustness in aa-tRNA accommodation despite gate defects.

Conclusions:

  • The ribosome's accommodation gate is not essential for rapid aa-tRNA entry and peptide bond formation.
  • Peptide release by RF2 is more sensitive to mutations in the accommodation gate region than aa-tRNA accommodation.
  • This suggests distinct mechanisms or sensitivities for aa-tRNA accommodation and RF2-mediated termination.

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