Transcription errors have been proposed to contribute to tumorigenesis and neurodegenerative diseases. An experimental validation of the biological impact of transcription errors faces major challenges, including the transient nature of mRNAs and the lack of a reliable methodology to distinguish transcription errors from translation errors. Here we report a genetic screen using Saccharomyces cerevisiae in which a transient transcription error is converted to a selectable genetic change. This assay can detect a transcription error rate as low as 1 × 10‐6. In this screen, we identified nine mutations in the catalytic subunit of RNA polymerase II (Pol II) that increased transcription errors in vivo. Functional in vitro analysis revealed that these mutations promoted misincorporation, improved mismatch extension, or decreased Pol II sensitivity to the proofreading factor TFIIS. Thus, we identify multiple molecular mechanisms that ensure that DNA is accurately transcribed in vivo. The error‐prone Pol II mutants will be used to elucidate the consequences of reduced transcription fidelity in yeast and mammalian cells and their role in human diseases. Grant Funding Source: Supported by NIH Intramural Research Program

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