RNA polymerase II mutants of RPB1 and RPB2 isolated using a novel assay for transcription fidelity in Saccharomyces cerevisiae

Thursday, October 11, 2012 — Poster Session IV
2:00 p.m. – 4:00 p.m.	Natcher Conference Center, Building 45	NCI	TRANS-1

Authors

• AS Denney
• DR Gotte
• JD Irvin
• KM Scibelli
• LL Lubkowska
• JN Strathern

Abstract

Transcription fidelity in vivo has long proved difficult to investigate since transcription errors are transient unlike permanent DNA events. Here we demonstrate the results of a novel assay whereby a transient transcription error is converted to a permanent genetic event and color phenotype in yeast colonies. We have designed a reporter gene as a target for Cre recombinase such that Cre restores the function of ADE6 and allows cells to produce a red pigment. Transcription slippage over a homopolymeric track of adenines upstream of the out-of-frame cre has the potential to correct its reading frame so that functional Cre is produced. We have used this system to screen for mutants with elevated transcription slippage as evidenced by increased red sectoring in colonies. Previously we have shown that this assay demonstrates significant phenotypes with known slippage-prone RNA polymerase II mutants of the two largest subunits, RPB1 and RPB2, relative to wild type. Here we describe the isolation and functional characterization of numerous mutations important for transcription slippage. Transcription fidelity is a vital cell process yet the consequences of transcriptional infidelity remain to be fully understood. Ultimately, we hope to contribute to this understanding as it relates to overall cellular health.

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