Vibrio harveyi RNA polymerase: purification and resolution from gyrase A

RNA polymerase was purified from Vibrio harveyi and found to contain polypeptides (β,β′, α, and σ) closely corresponding to those of the Escherichia coli enzyme. In vitro transcription studies using V. harveyi and E. coli RNA polymerase demonstrated that the purified V. harveyi RNA polymerase is functional and that the two enzymes have the same promoter specificity. Chromatography through a monoQ column was required to remove a 100-kilodalton protein that was present in large amounts and copurified with the RNA polymerase. N-terminal amino acid sequencing showed that the first 18 amino acids of the 100-kilodalton protein shares 78% sequence identity with the A subunit of gyrase or topoisomerase II. The abundance of the gyrase A protein is unprecedented and may be linked to bioluminescence.Key words: Vibrio harveyi, RNA polymerase, gyrase, bioluminescence.

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The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70 Subunit of Escherichia coli RNA Polymerase

Journal of Bacteriology ◽

10.1128/jb.184.14.3957-3964.2002 ◽

2002 ◽

Vol 184 (14) ◽

pp. 3957-3964 ◽

Cited By ~ 43

Author(s):

Suchira Pande ◽

Anna Makela ◽

Simon L. Dove ◽

Bryce E. Nickels ◽

Ann Hochschild ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Bacteriophage T4 ◽

In Vitro Transcription ◽

Terminal Region ◽

Transcription Activator ◽

E Coli ◽

Terminal Amino ◽

Two Hybrid

ABSTRACT Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the σ70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at −30, and also interacts with σ70. We show here that the N-terminal half of MotA (MotANTD), which is thought to include the activation domain, interacts with the C-terminal region of σ70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of σ70 with comparable σ38 residues abolishes the interaction with MotANTD in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a σ70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotACTD) binds DNA with a K D(app) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of σ70 helps to substitute functionally for an interaction between σ70 and a promoter −35 element.

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Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

Journal of Bacteriology ◽

10.1128/jb.183.7.2289-2297.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2289-2297 ◽

Cited By ~ 8

Author(s):

Marco P. Cicero ◽

Meghan M. Sharp ◽

Carol A. Gross ◽

Kenneth N. Kreuzer

Keyword(s):

Rna Polymerase ◽

Burst Size ◽

Bacteriophage T4 ◽

In Vitro Transcription ◽

Positive Control ◽

Plaque Morphology ◽

Mutant Proteins ◽

E Coli

ABSTRACT 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 ς70 subunit. AmotA 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 ς70 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 ς70 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 ς70 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. colipromoters. 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.

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A Positive cis-Acting DNA Element Is Required for High-Level Transcription in Chlamydia

Journal of Bacteriology ◽

10.1128/jb.182.18.5167-5171.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5167-5171 ◽

Cited By ~ 11

Author(s):

Chris S. Schaumburg ◽

Ming Tan

Keyword(s):

Escherichia Coli ◽

Chlamydia Trachomatis ◽

Rna Polymerase ◽

Promoter Region ◽

In Vitro Transcription ◽

Transcription Assay ◽

Cis Acting ◽

E Coli ◽

High Level

ABSTRACT The spacer A/T region is a positive cis-acting DNA element that was identified in the Chlamydia trachomatisrRNA promoter region. We have now demonstrated that similar sequences in other chlamydial promoters are important for transcription. Substitution of candidate spacer A/T regions in four chlamydial promoters decreased transcription by partially purified C. trachomatis RNA polymerase in an in vitro transcription assay. Addition of a spacer A/T region to the dnaK promoter, which does not contain an identifiable spacer A/T region, increased transcription 16-fold. Transcription of Escherichia colipromoters by C. trachomatis RNA polymerase also appeared to be dependent on the spacer A/T region. However, the effect of the spacer A/T region on transcription by E. coli RNA polymerase was small. In summary, the spacer A/T region is a novel DNA element that is required for high-level transcription of many promoters by chlamydial RNA polymerase.

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Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

Journal of Bacteriology ◽

10.1128/jb.180.9.2359-2366.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2359-2366 ◽

Cited By ~ 26

Author(s):

Ming Tan ◽

Tamas Gaal ◽

Richard L. Gourse ◽

Joanne N. Engel

Keyword(s):

Escherichia Coli ◽

Chlamydia Trachomatis ◽

Rna Polymerase ◽

Mutational Analysis ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Rich Region ◽

E Coli ◽

Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

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