title: A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.
abstract: 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.
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America; U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland, United States of America.
authors.forename: Jordan D
authors.initials: JD
authors.lastname: Irvin
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Kireeva
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Deanna R
authors.initials: DR
authors.lastname: Gotte
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Brenda K
authors.initials: BK
authors.lastname: Shafer
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Ingold
authors.initials: I
authors.lastname: Huang
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
authors.affiliation: NCI Center for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Strathern
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D003062:Codon / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D003062:Codon; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II