A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.
PLoS Genet
; 10(9): e1004532, 2014 Sep.
Article
em En
| MEDLINE
| ID: mdl-25232834
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.
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Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Transcrição Gênica
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RNA Polimerase II
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Proteínas de Saccharomyces cerevisiae
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article