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Related Experiment Videos

Missense translation errors in Saccharomyces cerevisiae

I Stansfield1, K M Jones, P Herbert

  • 1Research School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.

Journal of Molecular Biology
|September 12, 1998
PubMed
Summary

We developed a novel yeast assay to measure amino acid misincorporation during protein synthesis. This tool confirmed the ribosome

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Protein synthesis fidelity is crucial for cellular function.
  • Mistranslation, or the incorrect incorporation of amino acids, can lead to dysfunctional proteins.
  • Accurate in vivo measurement of mistranslation events is challenging.

Purpose of the Study:

  • To develop a novel plasmid-based assay for measuring in vivo amino acid misincorporation frequency in Saccharomyces cerevisiae.
  • To quantify specific mistranslation events and assess the impact of translational error-inducing agents.
  • To investigate the role of ribosomal proteins in controlling missense errors.

Main Methods:

  • Development of a chloramphenicol acetyl transferase (CATIII) active site mutant assay in yeast.

Related Experiment Videos

  • Measurement of histidine misincorporation at a tyrosine codon (His195(CAC)-->Tyr195(UAC)) and an alanine codon (Ala195 GCU).
  • Assessment of paromomycin's effect on misincorporation frequencies and analysis of SUP44 and SUP46 mutants.

    • Main Results:

    • The assay accurately measured histidine misincorporation at a tyrosine codon at a frequency of 0.5x10(-5) in wild-type yeast.
    • Paromomycin increased this misincorporation frequency by 50-fold, indicating translational error induction.
    • Increased missense errors were observed in SUP44 and SUP46 mutants, implicating ribosomal proteins S4 and S9 in error control.

    Conclusions:

    • The developed assay provides the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell.
    • The eukaryotic ribosome plays a significant role in controlling missense errors, particularly those arising from non-cognate codon-anticodon interactions.
    • This study validates the importance of translational fidelity and provides a tool for further investigation into mistranslation mechanisms.