A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity
| Autor: | Jeffrey N. Strathern, Jordan D. Irvin, Mikhail Kashlev, Maria L. Kireeva, Brenda Shafer, Deanna Gotte, Ingold Huang |
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| Rok vydání: | 2014 |
| Předmět: |
Genetic Screens
Cancer Research Saccharomyces cerevisiae Proteins lcsh:QH426-470 Transcription Genetic Molecular Sequence Data DNA transcription Gene Identification and Analysis Cre recombinase RNA polymerase II Saccharomyces cerevisiae Biochemistry chemistry.chemical_compound Genes Reporter Transcription (biology) Catalytic Domain Gene Expression Regulation Fungal Nucleic Acids RNA polymerase Genetics Amino Acid Sequence RNA synthesis Codon Molecular Biology Gene Genetics (clinical) Ecology Evolution Behavior and Systematics Biology and life sciences biology Point mutation Intron RNA stability lcsh:Genetics chemistry Mutation biology.protein RNA Molecular Complexes RNA Polymerase II Gene expression Research Article Genetic screen |
| Zdroj: | PLoS Genetics PLoS Genetics, Vol 10, Iss 9, p e1004532 (2014) |
| ISSN: | 1553-7404 |
| DOI: | 10.1371/journal.pgen.1004532 |
| Popis: | We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing. Author Summary Mistakes made during the synthesis of messenger RNAs have been difficult to detect, both because mRNAs can be short lived, and because the translation of mRNAs into proteins has a much higher error rate that masks transcription errors. We present here a highly sensitive genetic screen that detects transcription errors and use it to identify mutations that increase the error rate of RNA polymerase II. The screen incorporates a new principle that allows transient transcription errors to cause permanent genetic changes. The screen is based on suppression of a missense mutation (cre-Y324C) in the active site of the Cre recombinase. Infrequent and transient transcription errors that restore the original codon for Y324 cause the Cre-dependent activation of a reporter gene. Background from translation errors is negligible because Cre acts as a tetramer in which all four subunits require the active site tyrosine. Transcription errors as low as ∼10−6 can be detected. We identify rpb1 mutations that define four classes, those that have increased (1) misincorporation, (2) extension of a misincorporated base, (3) both misincorporation and extension, and (4) those that block the activity of the transcription proofreading factor, TFIIS. |
| Databáze: | OpenAIRE |
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