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Journal of Bacteriology, April 2001, p. 2289-2297, Vol. 183, No. 7
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.7.2289-2297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Substitutions in Bacteriophage T4 AsiA and Escherichia coli sigma 70 That Suppress T4 motA Activation Mutations

Marco P. Cicero,1,dagger Meghan M. Sharp,2 Carol A. Gross,2 and Kenneth N. Kreuzer1,*

Departments of Microbiology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710,1 and Department of Stomatology, University of California at San Francisco, San Francisco, California 941432

Received 10 October 2000/Accepted 19 January 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.

* Corresponding author. Mailing address: Department of Microbiology, Duke University Medical Center, Box 3020, Durham NC 27710. Phone: (919) 684-6466. Fax: (919) 681-8911. E-mail: kenneth.kreuzer{at}duke.edu.

dagger Present address: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059.

Journal of Bacteriology, April 2001, p. 2289-2297, Vol. 183, No. 7
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.7.2289-2297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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