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

Shwu Yuan Wu, Terry Platt

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 II as well as for correct 3′-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species. Transcription in our processing-proficient 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. coli rho-dependent termination signal trp 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 trp t′ template was used affirmed that they were due to the effects of rho. E. coli RNA polymerase added to a yeast extract pretreated with α-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 II. Thus, functional rho and its cognate site, trp t′, appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination.

Original languageEnglish (US)
Pages (from-to)6606-6610
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number14
StatePublished - Jul 15 1993

Fingerprint

RNA Polymerase II
Escherichia coli Proteins
Yeasts
Escherichia coli
Cell Extracts
RNA 3' End Processing
RNA
Amanitins
DNA-Directed RNA Polymerases
Saccharomyces cerevisiae
Proteins
Nucleotides
In Vitro Techniques
DNA
Enzymes

Keywords

  • RNA 3′ ends
  • RNA processing
  • Transcription termination

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

@article{941fc2b76ac04ec79f8c39c6aeb3fcc8,
title = "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 II as well as for correct 3′-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species. Transcription in our processing-proficient 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. coli rho-dependent termination signal trp 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 trp t′ template was used affirmed that they were due to the effects of rho. E. coli RNA polymerase added to a yeast extract pretreated with α-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 II. Thus, functional rho and its cognate site, trp t′, appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination.",
keywords = "RNA 3′ ends, RNA processing, Transcription termination",
author = "Wu, {Shwu Yuan} and Terry Platt",
year = "1993",
month = "7",
day = "15",
language = "English (US)",
volume = "90",
pages = "6606--6610",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "14",

}

TY - JOUR

T1 - Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro

AU - Wu, Shwu Yuan

AU - Platt, Terry

PY - 1993/7/15

Y1 - 1993/7/15

N2 - 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 II as well as for correct 3′-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species. Transcription in our processing-proficient 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. coli rho-dependent termination signal trp 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 trp t′ template was used affirmed that they were due to the effects of rho. E. coli RNA polymerase added to a yeast extract pretreated with α-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 II. Thus, functional rho and its cognate site, trp t′, appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination.

AB - 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 II as well as for correct 3′-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species. Transcription in our processing-proficient 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. coli rho-dependent termination signal trp 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 trp t′ template was used affirmed that they were due to the effects of rho. E. coli RNA polymerase added to a yeast extract pretreated with α-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 II. Thus, functional rho and its cognate site, trp t′, appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination.

KW - RNA 3′ ends

KW - RNA processing

KW - Transcription termination

UR - http://www.scopus.com/inward/record.url?scp=0027184488&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027184488&partnerID=8YFLogxK

M3 - Article

C2 - 8341675

AN - SCOPUS:0027184488

VL - 90

SP - 6606

EP - 6610

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 14

ER -