Isolation and characterization of transcription fidelity mutants

Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

► Accurate RNA synthesis is necessary for preservation of biological information. ► Studying transcription fidelity is a challenge due to the transient nature of mRNAs. ► Genetic schemes for the isolation of fidelity RNA polymerase mutants are developed. ► Biochemical assays for detection of transcription errors are designed. ► The consequences of transcription errors by the fidelity mutants are discussed.