Repository logo

Infoscience

  • English
  • French
Log In
Logo EPFL, École polytechnique fédérale de Lausanne

Infoscience

  • English
  • French
Log In
  1. Home
  2. Academic and Research Output
  3. Journal articles
  4. Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase.
 
Loading...
Thumbnail Image
research article

Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase.

Lee, J Y
•
Zalenskaya, K
•
Shin, Y K
Show more
1989
Journal of bacteriology

The rifampin resistance rifD18 allele of rpoB, carried on the expression plasmid pXT7 beta, is controlled by a strong bacteriophage T7 late promoter and two weak Escherichia coli promoters. Depending on the host strain, pXT7 beta specifies different levels of Rifr beta subunit, providing a system for the isolation, maintenance, and overexpression of dominant lethal alleles of rpoB. In rpoB+ hosts, pXT7 beta confers the Rifr phenotype on the Rifs host. Negative rpoB mutations in the plasmid DNA can thus be scored by screening transformants for Rifs. In an rpoB(Am) supD(Ts) host in which chromosomal rpoB expression is decreased as the temperature goes up, some of the negative plasmid-borne rpoB mutations displayed a dominant phenotype. In a host harboring inducible T7 RNA polymerase, the defective beta subunits could be overexpressed independently of the E. coli transcriptional machinery. With this system, we isolated several negative rpoB mutations induced in vitro by hydroxylamine. Seven of the mutant rpoB alleles, when overexpressed, were found to specify normal-size beta polypeptides. Two of them displayed the dominant lethal phenotype in the rpoB(Am) supD(Ts) background. We also constructed a mutation (rpoB1800) in which 24 carboxy-terminal amino acids were substituted with a random 19-amino-acid sequence. The nonfunctional rpoB1800 beta polypeptide was isolated and assembled in vitro into the core enzyme molecule.

  • Details
  • Metrics
Type
research article
DOI
10.1128/jb.171.6.3002-3007.1989
PubMed ID

2656636

Author(s)
Lee, J Y
•
Zalenskaya, K
•
Shin, Y K
•
McKinney, J D  
•
Park, J H
•
Goldfarb, A
Date Issued

1989

Published in
Journal of bacteriology
Volume

171

Issue

6

Start page

3002

End page

7

Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
UPKIN  
Available on Infoscience
September 7, 2010
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/52837
Logo EPFL, École polytechnique fédérale de Lausanne
  • Contact
  • infoscience@epfl.ch

  • Follow us on Facebook
  • Follow us on Instagram
  • Follow us on LinkedIn
  • Follow us on X
  • Follow us on Youtube
AccessibilityLegal noticePrivacy policyCookie settingsEnd User AgreementGet helpFeedback

Infoscience is a service managed and provided by the Library and IT Services of EPFL. © EPFL, tous droits réservés