Bacillus subtilis σB is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase

Andrew K. Benson, William G. Haldenwang

Research output: Contribution to journalArticle

149 Citations (Scopus)

Abstract

σB is a secondary σ factor of Bacillus subtilis. RNA polymerase containing σB transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the σB structural gene (sigB), regulate σB-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the σB regulatory pathway. Evidence is now presented that RsbW inhibits σB-dependent transcription by binding to σB and blocking the formation of a σB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or σB will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of σB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified σB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a σB-dependent promoter (etc) in vitro. RsbW efficiently blocked σB-dependent transcription but only if it was incubated with σB prior to the addition of the core enzyme.

Original languageEnglish (US)
Pages (from-to)2330-2334
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number6
DOIs
StatePublished - Mar 15 1993

Fingerprint

DNA-Directed RNA Polymerases
Bacillus subtilis
Carrier Proteins
RNA Polymerase II
Genes
Holoenzymes
Cell Extracts
Complex Mixtures
Gel Chromatography
Shock
Proteins
Hot Temperature
Western Blotting
Gene Expression
Antibodies
Enzymes
Growth

ASJC Scopus subject areas

  • General

Cite this

@article{4ddb8ddf57794590a429b48e2f0db668,
title = "Bacillus subtilis σB is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase",
abstract = "σB is a secondary σ factor of Bacillus subtilis. RNA polymerase containing σB transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the σB structural gene (sigB), regulate σB-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the σB regulatory pathway. Evidence is now presented that RsbW inhibits σB-dependent transcription by binding to σB and blocking the formation of a σB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or σB will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of σB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified σB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a σB-dependent promoter (etc) in vitro. RsbW efficiently blocked σB-dependent transcription but only if it was incubated with σB prior to the addition of the core enzyme.",
author = "Benson, {Andrew K.} and Haldenwang, {William G.}",
year = "1993",
month = "3",
day = "15",
doi = "10.1073/pnas.90.6.2330",
language = "English (US)",
volume = "90",
pages = "2330--2334",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "6",

}

TY - JOUR

T1 - Bacillus subtilis σB is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase

AU - Benson, Andrew K.

AU - Haldenwang, William G.

PY - 1993/3/15

Y1 - 1993/3/15

N2 - σB is a secondary σ factor of Bacillus subtilis. RNA polymerase containing σB transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the σB structural gene (sigB), regulate σB-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the σB regulatory pathway. Evidence is now presented that RsbW inhibits σB-dependent transcription by binding to σB and blocking the formation of a σB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or σB will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of σB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified σB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a σB-dependent promoter (etc) in vitro. RsbW efficiently blocked σB-dependent transcription but only if it was incubated with σB prior to the addition of the core enzyme.

AB - σB is a secondary σ factor of Bacillus subtilis. RNA polymerase containing σB transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the σB structural gene (sigB), regulate σB-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the σB regulatory pathway. Evidence is now presented that RsbW inhibits σB-dependent transcription by binding to σB and blocking the formation of a σB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or σB will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of σB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified σB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a σB-dependent promoter (etc) in vitro. RsbW efficiently blocked σB-dependent transcription but only if it was incubated with σB prior to the addition of the core enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0027401048&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027401048&partnerID=8YFLogxK

U2 - 10.1073/pnas.90.6.2330

DO - 10.1073/pnas.90.6.2330

M3 - Article

C2 - 8460143

AN - SCOPUS:0027401048

VL - 90

SP - 2330

EP - 2334

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 - 6

ER -