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Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations.

Publication ,  Journal Article
Cicero, MP; Sharp, MM; Gross, CA; Kreuzer, KN
Published in: J Bacteriol
April 2001

Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the sigma(70) subunit. A motA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five sigma(70) mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant sigma(70) proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The sigma(70) substitutions affected the 4.2 region, which binds the -35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. coli promoters. This defect could not be explained solely by a disruption in -35 recognition since similar results were obtained with extended -10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

Duke Scholars

Published In

J Bacteriol

DOI

ISSN

0021-9193

Publication Date

April 2001

Volume

183

Issue

7

Start / End Page

2289 / 2297

Location

United States

Related Subject Headings

  • Viral Proteins
  • Transcription, Genetic
  • Transcription Factors
  • Sigma Factor
  • Promoter Regions, Genetic
  • Mutation
  • Microbiology
  • Escherichia coli
  • DNA-Directed RNA Polymerases
  • DNA-Binding Proteins
 

Citation

APA
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ICMJE
MLA
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Cicero, M. P., Sharp, M. M., Gross, C. A., & Kreuzer, K. N. (2001). Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations. J Bacteriol, 183(7), 2289–2297. https://doi.org/10.1128/JB.183.7.2289-2297.2001
Cicero, M. P., M. M. Sharp, C. A. Gross, and K. N. Kreuzer. “Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations.J Bacteriol 183, no. 7 (April 2001): 2289–97. https://doi.org/10.1128/JB.183.7.2289-2297.2001.
Cicero MP, Sharp MM, Gross CA, Kreuzer KN. Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations. J Bacteriol. 2001 Apr;183(7):2289–97.
Cicero, M. P., et al. “Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations.J Bacteriol, vol. 183, no. 7, Apr. 2001, pp. 2289–97. Pubmed, doi:10.1128/JB.183.7.2289-2297.2001.
Cicero MP, Sharp MM, Gross CA, Kreuzer KN. Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations. J Bacteriol. 2001 Apr;183(7):2289–2297.

Published In

J Bacteriol

DOI

ISSN

0021-9193

Publication Date

April 2001

Volume

183

Issue

7

Start / End Page

2289 / 2297

Location

United States

Related Subject Headings

  • Viral Proteins
  • Transcription, Genetic
  • Transcription Factors
  • Sigma Factor
  • Promoter Regions, Genetic
  • Mutation
  • Microbiology
  • Escherichia coli
  • DNA-Directed RNA Polymerases
  • DNA-Binding Proteins