Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.

Abstract

A promoter-independent assay utilizing poly(dC)-tailed DNA templates has revealed that Saccharomyces cerevisiae whole-cell extracts can be proficient for transcription by the endogenous yeast RNA polymerase H as well as for correct 3'-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace btanscription products corresponded to the expected size of terminated RNA species. Transcription in our processingproficient extract was thus insufficient to cause termination. To test our system with a known, albeit heterologous, signal, we examined a dC-tailed template carrying the E. col rhodependent termination signal £p t' in the yeast extract. Transcripts from this template were not susceptible to processing, but addition of rho protein resulted in two distinct truncated transcripts that could not be chased by excess unlabeled nucleotides. These RNA species thus represented stably paused or terminated polymerase II products, and their absence when a mutated unresponsive tip t' template was used affirmed that they were due to the effects of rho. E. col RNA polymerase added to a yeast extract pretreated with a-amanitin was also halted by rho at these same two sites. A mutated rho protein, while only partly defective with E. coli polymerase, failed to provoke arrest when transcription was carried out by RNA polymerase H. Thus, functional rho and its cognate site, tip t', appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast wholecell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase H transcription termination.Transcription termination is required to complete RNA synthesis and prevent transcriptional interference with downstream genes. It occurs when the RNA polymerase dissociates from the DNA template and the nascent RNA is released. Termination mechanisms are classified into two major categories (1). Intrinsic termination occurs as RNA polymerase stops in direct response to signals encoded in the RNA transcript and/or the DNA template. Alternative mechanisms require the action of some additional trans-acting factor(s) to terminate RNA elogation by the polymerase molecules. Among identified bacterial termination factors, the involvement of Escherichia coli rho protein in transcription termination is best understood (2).In eukaryotes, the study of RNA polymerase II (pol II) termination is complicated by 3'-end RNA processing, which produces mature mRNA 3' ends via specific endonucleolytic cleavage and polyadenylylation. To distinguish between termination and RNA processing events, nuclear run-on studies in vivo or transcription experiments in vitro are required. However, the former are tedious and time-consuming, and the latter are difficult due to the complexities of pol II The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

Transcription termination and anti‐termination in E. coli

“…Alternatively, Rho might use ATP hydrolysis coupled with the threading motion in its cavity to pull RNA out of the HBS, independently of its helicase activity. This latter possibility does not entail specific contacts between Rho and RNAP, consistent with the ability of Rho to terminate the transcription of non‐bacterial RNAP, such as yeast RNAP II (Wu & Platt 1993; Lang et al . 1998) and the RNAPs of phage T7 or SP6 (Pasman & von Hippel 2000).…”

Regulation of Transcription Elongation and Termination

“…Continued pressure then exposes the transcription hybrid to Rho either by a conformational change in RNAP or by removal without forward translocation. Termination may not entail specific interactions with RNAP, since E. coli Rho factor will efficiently terminate transcription of Saccharomyces cerevisiae RNA pol II [ 26 ].…”

Rho and RNA: models for recognition and response

“…What is the role of kinetic coupling (Jin et ai. 1992), and does Rho interact directly with RNA polymerase(s), as suggested circumstantially by many observations, including its ability to arrest yeast RNA poiymerase II (Wu and Platt, 1993)? Is Rho's helicase activity employed to strip the transcript out of a cationic binding site on the poiymerase core molecule instead of (or in addition to) disrupting RNA-template pairing, as suggested by Surratt et ai (1991)?…”