Related Experiment Videos
Yeast tRNATrp genes with anticodons corresponding to UAA and UGA nonsense codons
D Kim1, G J Raymond, S D Clark
1Department of Molecular Biology, University of Wyoming, Laramie 82071.
Abstract:
Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.
Insights
Researchers created ochre and opal suppressor mutations in yeast tRNATrp genes. The opal suppressor showed moderate activity at high copy numbers, while the ochre suppressor remained inactive, impacting tRNA levels.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- Naturally occurring suppressor mutants of tRNATrp genes are unknown in Saccharomyces cerevisiae.
- Oligonucleotide-directed mutagenesis is a key tool for generating specific genetic alterations.
Purpose of the Study:
- To engineer ochre and opal suppressor mutations into the S. cerevisiae tRNATrp gene.
- To assess the in vitro and in vivo suppressor activity and tRNA accumulation of these engineered genes.
Main Methods:
- Site-directed mutagenesis was used to create ochre (TRPO) and opal (TRPOP) suppressor variants of the tRNATrp gene.
- In vitro transcription, processing, and splicing assays were performed.
- Engineered genes were introduced into yeast strains with various nonsense mutations using CEN and high copy-number vectors.
- Northern blot analysis was used to quantify tRNA levels.
Main Results:
- Both TRPO and TRPOP variants were transcribed, processed, and spliced in vitro.
- TRPO accumulated precursors and tRNA comparably to the wild-type, while TRPOP accumulated significantly less tRNA.
- Neither suppressor showed activity on a low-copy CEN vector.
- The TRPOP variant exhibited moderate suppressor activity on a high copy-number vector, whereas TRPO showed no detectable activity.
- High copy-number clones led to 20-100% increases in tRNATrp steady-state levels.
Conclusions:
- Engineered ochre and opal suppressor tRNATrp genes can be created in S. cerevisiae.
- Suppressor activity is dependent on gene copy number, with the opal suppressor showing partial function at high expression levels.
- The ochre suppressor variant lacks detectable biological activity, suggesting limitations in its ability to suppress nonsense mutations in yeast.