Polypeptides containing highly conserved regions of transcription initiation factor σ70 exhibit specificity of binding to promoter DNA

Dombroski, Alicia J.; Walter, William; Record, M. Thomas; Slegele, Deborah A.; Gross, Carol A.

doi:10.1016/0092-8674(92)90174-b

Cited by 313 publications

(320 citation statements)

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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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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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“…ES-MS, electrospray mass spectrometry ; IPTG, isopropyl thio-P-D-galactoside ; NTP, nucleoside triphosphate ; TRES, timeresolved emission spectroscopy. nition, respectively (Gardella et al, 1989;Siegele et al, 1989;Waldburger et al, 1990), are masked by the N-terminal domain of 07", thereby preventing free 0 7 0 from interacting with promoter DNA (Dombroski et al, 1992). 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.…”

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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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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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“…In the absence of core RNAP, 70 -like factors are unable to recognize promoter DNA in either double-or single-stranded form (11). Specific interactions between N-terminally truncated derivatives of 70 and promoter DNA were detected by using competitive filter-binding assays (11,12), leading to the hypothesis that the latent DNA-binding activity of is inhibited by the N-terminal region 1.1, and that this inhibition is relieved by conformational changes upon binding of to core RNAP. Studies of isolated 70 fragments revealed that region 1.1 inhibited region 4.2 binding to the Ϫ35 element in trans, but not region 2 binding to the Ϫ10 element.…”

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confidence: 99%

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Camarero

Shekhtman

Campbell

et al. 2002

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

115

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Bacterial factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequencespecific interactions with the ؊10 and ؊35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the ؊35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a 70 -like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and͞or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.T he 400-kDa bacterial core RNA polymerase (RNAP) is fully active in RNA polymerization but is incapable of promoter recognition and specific transcription initiation. Promoter recognition, promoter melting to form the open complex, and possibly other functions during transcription initiation, depend on the binding of a factor to the core RNAP (subunit composition ␣ 2 ␤␤Ј ) to form the RNAP holoenzyme (reviewed in ref. 1). The primary factor in Escherichia coli, responsible for the bulk of transcription during exponential growth, is 70 . Sequence comparisons reveal that 70 belongs to a large homologous family of proteins with four regions of highly conserved amino acid sequence (2-5) (Fig. 1).The factors direct the process of transcription initiation by first locating the promoter through sequence-specific recognition of two hexamers of consensus DNA; the Pribnow box or Ϫ10 element, centered at about Ϫ10 with respect to the transcription start site (ϩ1), and the Ϫ35 element (6, 7). Genetic and biophysical evidence strongly suggested that amino acid residues in region 4.2 recognize the Ϫ35 element (8, 9), and this was confirmed by structural studies (10). Nonetheless, most functions of , including sequence-specific DNA binding, manifest themselves only in the context of the RNAP holoenzyme. In the absence of core RNAP, 70 -like factors are unable to recognize promoter DNA in either double-or single-stranded form (11). Specific interactions between N-terminally truncated derivatives of 70 and promoter DNA were detected by using competitive filter-binding assays (11,12), leading to the hypothesis that the latent DNA-binding activity of is inhibited by the N-terminal region 1.1, and that this inhibition is relieved by conformational changes upon binding of to core RNAP. Studies of isolated 70 fragments revealed that region 1.1 inhibited region 4.2 binding to the Ϫ35 element in trans, but not region 2 binding to the Ϫ10 element. These results led to a more detailed model in which region 1.1 directly masks the DNA-binding determinants of region 4.2 (11, 12). I...

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Polypeptides containing highly conserved regions of transcription initiation factor σ70 exhibit specificity of binding to promoter DNA

Cited by 313 publications

References 48 publications

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

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

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

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

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Polypeptides containing highly conserved regions of transcription initiation factor σ70 exhibit specificity of binding to promoter DNA

Cited by 313 publications

References 48 publications

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

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

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

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

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

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

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