Related Experiment Videos
MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase
Abstract:
E569 and E606 are noncomplementing pet mutants of Saccharomyces cerevisiae. Both strains are defective in mitochondrial protein synthesis and as a result exhibit a pleiotropic deficiency in respiratory components that are translated on mitochondrial ribosomes. The wild type gene MSW capable of complementing the protein synthesis defect has been cloned by transformation of one of the mutants with a genomic library of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein with a molecular weight of 42,414 which is 37 and 39% identical to the tryptophanyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus, respectively. A strain containing an insertion in the chromosomal copy of MSW was constructed by in situ gene replacement. This mutant fails to charge mitochondrial tryptophanyl-tRNA providing further evidence that MSW is the structural gene for mitochondrial tryptophanyl tRNA synthetase. The existence of another gene coding for the cytoplasmic tryptophanyl-tRNA synthetase is inferred from the observation that mutations in MSW are not lethal but only result in a respiratory deficiency.
Insights
Researchers identified the MSW gene in Saccharomyces cerevisiae, crucial for mitochondrial protein synthesis. This gene encodes mitochondrial tryptophanyl-tRNA synthetase, essential for respiratory function.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Mitochondrial Biology
Background:
- Saccharomyces cerevisiae mutants E569 and E606 exhibit defects in mitochondrial protein synthesis.
- These defects lead to deficiencies in respiratory components synthesized by mitochondrial ribosomes.
Purpose of the Study:
- To clone and characterize the gene responsible for complementing the mitochondrial protein synthesis defect in yeast mutants.
- To identify the specific function of the cloned gene in mitochondrial processes.
Main Methods:
- Cloning the wild-type MSW gene by yeast transformation using a genomic library.
- DNA sequencing of the cloned gene to determine its coding potential.
- Construction of a knockout mutant using in situ gene replacement to confirm gene function.
Main Results:
- The cloned gene, MSW, complements the mitochondrial protein synthesis defect.
- MSW encodes a protein homologous to tryptophanyl-tRNA synthetases from E. coli and B. stearothermophilus.
- A knockout mutant lacking MSW fails to charge mitochondrial tryptophanyl-tRNA, confirming MSW as the structural gene.
Conclusions:
- MSW is the structural gene for mitochondrial tryptophanyl-tRNA synthetase in Saccharomyces cerevisiae.
- Mitochondrial tryptophanyl-tRNA synthetase is essential for respiratory deficiency.
- The existence of a separate gene for cytoplasmic tryptophanyl-tRNA synthetase is implied.