Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Dombroski, Alicia J.; Walter, William; Gross, Carol A.

doi:10.1101/gad.7.12a.2446

Cited by 207 publications

(213 citation statements)

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“…We d o not want to emphasize the p-structure (Table 1) that was generated in the mutant protein as any estimation from very low intense CD bands is susceptible to error. Fluorescence studies with l-anilinonapthalene-8-sulphonate, however, indicate that both N-terminal and C-terminal mutations in 07" expose similar hydrophobic surfaces for the probe and they were buried in native 07 '. We have mentioned before that interaction of the N-terminal portion of 07" with the promoter-binding regions at the C-terminal end is responsible for masking the DNA-binding ability of free a7" (Dombroski et al, 1993). We proposed a model earlier where it was speculated that Trp434 at the junction of the 2.31 2.4 subdomain in 0 " interacts with the hydrophobic pockets at the N-terminal domain (Gopal e t al., 1994).…”

Section: Discussionmentioning

confidence: 99%

“…Upon binding core RNA polymerase, the DNA-binding domains in a are unmasked and the resulting holoenzyme recognizes the promoter sequence with the help of 07" to initiate promoter-specific transcription. Dombroski et al (1993) showed that the N-terminal 1.1 subdomain of 07" preferentially inhibits DNA binding by domain 4 of 07" in trans, in comparison to that by domain 2.…”

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confidence: 99%

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Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

Gopal

Chatterji

1997

European Journal of Biochemistry

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Among various group I sigma factors, two amino acids, Val55 and Ala59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]o7 ' and [Gly59]070, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type 0 7 0 has an apparent molecular mass of 87 kDa. However, [Gly434]07", which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]07" and [Gly59]o7O have reduced helicity (20%) compared to wild-type 07" (50%). Binding of sigma factors with the hydrophobic, surface active probe 1 -anilinonapthalene-8-sulphonate, has shown that more hydrophobic surfaces are available/exposed in [Gly55]07', [Gly59]a7" as well as in [Gly434]a7" in comparison to wild-type 07". Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (a2PF) have shown reduced transcriptional activity in comparison to the enzyme containing wild-type sigma factor. However, another mutation (Val+Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83]a7", has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in 0 ' " may interact hydrophobically with the 2.3/2.4 DNA-binding region.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

Gopal

Chatterji

1997

European Journal of Biochemistry

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“…Discussion Region 1.1, the negatively-charged domain found at the N terminus of primary factors, such as 70 , is known to serve several important roles. In free 70 , the presence of this region prevents recognition of promoter DNA (53,54). In holoenzyme, region 1.1 lies within a channel of core polymerase that will interact with dsDNA downstream of the transcription start site upon formation of the stable promoter/polymerase complex (36).…”

Section: Figmentioning

confidence: 99%

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

Hook-Barnard

Hinton

2009

Proc. Natl. Acad. Sci. U.S.A.

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Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of 70 , is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P minor promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P minor spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P minor derivative that contains good 70 -dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P minor sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the Pminor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a ''gatekeeper'' to monitor channel entry.

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“…The transcription factors evolutionarily related to Escherichia coli 70 are subdivided into two groups (15). The primary factor is required for the expression of housekeeping genes whose products are essential for exponential cell growth.…”

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Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar

1997

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A homolog of the multiple-stress-responsive transcription factor B of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated SB , indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis B -dependent P B promoter of the sigB operon was stimulated by SB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted B -dependent P B promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary factor SA , while that of the P3 promoter is dependent on SB . As determined by primer extension studies, the 5 end of the SB -initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5 end of the cellular RNA.In prokaryotes, several factors coexist in cells (19,22,24). The transcription factors evolutionarily related to Escherichia coli 70 are subdivided into two groups (15). The primary factor is required for the expression of housekeeping genes whose products are essential for exponential cell growth. The level or the activity of the second group, comprising the alternate factors, has been shown to be modulated in response to environmental and metabolic stresses. The core RNA polymerase (RNAP) associated with a particular factor initiates transcription from a specific set of promoters with conserved sequence motifs. B , identified in sporulating Bacillus subtilis, is the first member of the alternate factors to be reported in prokaryotes (20,21). Much has been learnt recently about the regulation of expression of the sigB operon and the modulation of the function of the SigB ( B ) protein in response to changing stress (entry into stationary growth phase, nutrient depletion, and presence of uncouplers of oxidative phosphorylation) and environmental stress (heat shock and osmotic shock) in B. subtilis (2,6,8,22,36,37,41).The emergence of multiple-drug-resistant, highly pathogenic strains of Staphylococcus aureus poses a serious thr...

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Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Cited by 207 publications

References 29 publications

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar

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Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Cited by 207 publications

References 29 publications

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ70 and Disruption of its Overall Structure

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ70 and Disruption of its Overall Structure

The promoter spacer influences transcription initiation via σ 70 region 1.1 of Escherichia coli RNA polymerase

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar

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Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ⁷⁰ and Disruption of its Overall Structure

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase