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High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes
09:44

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Published on: March 3, 2015

Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein

A L Benko1, G Vaduva, N C Martin

  • 1Department of Biochemistry, Pennsylvania State University College of Medicine, H171, 500 University Drive, Hershey, PA 17033, USA.

Proceedings of the National Academy of Sciences of the United States of America
|January 5, 2000
PubMed

Insights

Yeast cells use the Mod5 protein to modify tRNAs, essential for protein synthesis. Competition for a shared substrate between Mod5 and Erg20 proteins impacts tRNA modification and translation efficiency.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The Saccharomyces cerevisiae Mod5 protein is crucial for tRNA modification, catalyzing isopentenylation to i(6)A in various cellular compartments.
  • Dimethylallyl pyrophosphate serves as a substrate for both Mod5p and Erg20p, an enzyme vital for sterol biosynthesis.

Purpose of the Study:

  • To investigate the impact of Mod5 protein distribution on tRNA modification and tRNA-mediated nonsense suppression.
  • To identify genes that influence nonsense suppression when overexpressed.

Main Methods:

  • Development of a colony color/growth assay to evaluate tRNA-mediated nonsense suppression.
  • Screening for genes affecting nonsense suppression through overexpression.
  • Midwestern analysis to assess tRNA i(6)A levels in yeast cells overproducing Erg20p.

Main Results:

  • Overexpression of SAL6, TEF4, YDL219w, and ARC1 affected nonsense suppression, suggesting roles in protein synthesis machinery or aminoacyl-tRNA synthetase interaction.
  • Identification of ERG20 as a gene influencing nonsense suppression.
  • Yeast cells overproducing Erg20p exhibited reduced levels of tRNA i(6)A, indicating competition for dimethylallyl pyrophosphate.

Conclusions:

  • Erg20p competes with Mod5p for the shared substrate dimethylallyl pyrophosphate, thereby affecting tRNA modification and nonsense suppression.
  • The study reveals a limited pool of dimethylallyl pyrophosphate in yeast.
  • Imbalances in enzymes utilizing dimethylallyl pyrophosphate significantly impact translational processes.

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