A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity

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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.

Vyšlo v časopise: A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity. PLoS Genet 10(9): e32767. doi:10.1371/journal.pgen.1004532
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004532

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