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Yeast killer virus transcription initiation in vitro
F P Barbone1, T L Williams, M J Leibowitz
1University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Department of Molecular Genetics and Microbiology, Piscataway 08854-5635.
Virology
|March 1, 1992
Summary
Killer virions possess RNA polymerase activity, transcribing viral RNA. This enzyme
Area of Science:
- Virology
- Molecular Biology
- Yeast Genetics
Background:
- Killer yeasts like Saccharomyces cerevisiae harbor viruses with dsRNA genomes.
- These viruses produce killer toxins, mediated by viral RNA components.
Purpose of the Study:
- To characterize the RNA polymerase activity within killer virions.
- To investigate the in vitro transcription of viral genomic dsRNA segments.
- To assess the translational competence of synthesized RNA transcripts.
Main Methods:
- Isolation of killer virions from infected Saccharomyces cerevisiae.
- In vitro transcription assays using viral RNA polymerase and dsRNA templates (L-A and M segments).
- Initiation of transcription with gamma-thio-GTP and cap analog 7mGpppG.
- Analysis of transcript 5' end modification and translational activity.
Main Results:
- Virion-associated RNA polymerase activity was confirmed, transcribing positive-sense RNA.
- Transcription initiation with gamma-thio-GTP incorporated thiophosphate at the 5' terminus.
- Transcripts produced with 7mGpppG showed enhanced translational activity in vitro.
- Viral RNA polymerase transcripts with 7mGpppG did not bind to anti-7mG antibodies, unlike E. coli polymerase transcripts.
Conclusions:
- The killer virion RNA polymerase synthesizes functional viral RNA transcripts.
- The enzyme's ability to incorporate cap structures influences RNA translational efficiency.
- Viral RNA polymerase exhibits distinct capping properties compared to bacterial polymerases.