Disulfide cross-linking indicates that FlgM-bound and free σ
            <sup>28</sup>
            adopt similar conformations

Sörenson, Margareta K.; Darst, Seth A.

doi:10.1073/pnas.0606482103

Cited by 23 publications

(32 citation statements)

References 23 publications

(37 reference statements)

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“…The extensive interdomain contacts observed within σ 28 led to the proposal that the conformation of σ 28 observed in complex with FlgM is stable in solution even in the absence of FlgM, and this proposal was confirmed experimentally using a disulfide crosslinking approach [40]. These findings suggest that the DNA-binding determinants for all σ 70 -family members may be masked by interdomain contacts in free σ.…”

Section: Flagellar Biosynthesis and The σ 28 /Flgm Complexmentioning

confidence: 62%

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

Campbell

Westblade

Darst

2008

Current Opinion in Microbiology

126

119

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SummaryIn bacteria, σ factors are essential for the promoter DNA-binding specificity of RNA polymerase. The σ factors themselves are regulated by anti-σ factors that bind and inhibit their cognate σ factor, and 'appropriators' that deploy a particular σ-associated RNA polymerase to a specific promoter class. Adding to the complexity is the regulation of anti-σ factors by both anti-anti-σ factors, which turn on σ factor activity, and co-anti-σ factors that act in concert with their partner anti-σ factor to inhibit or redirect σ activity. While σ factor structure and function is highly conserved, recent results highlight the diversity of structures and mechanisms that bacteria use to regulate σ factor activity, reflecting the diversity of environmental cues that the bacterial transcription system has evolved to respond to.

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Section: Flagellar Biosynthesis and The σ 28 /Flgm Complexmentioning

confidence: 62%

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

Campbell

Westblade

Darst

2008

Current Opinion in Microbiology

126

119

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“…For D , structural analysis of a D ::FlgM complex revealed a compact conformation with the two DNA-binding domains (regions 2 and 4) closely apposed (26). Disulfide cross-linking suggests that a similarly compact conformation predominates in solution (25). Conversely, other studies support the idea that factors may specifically recognize elements of the promoter even in the absence of core RNA polymerase (12,16,23).…”

mentioning

confidence: 79%

“…However, many alternative factors lack region 1, and a different mechanism of self-inhibition likely pertains. Indeed, solution studies suggest a predominant conformation incompatible with DNA binding (22,25). For D , structural analysis of a D ::FlgM complex revealed a compact conformation with the two DNA-binding domains (regions 2 and 4) closely apposed (26).…”

mentioning

confidence: 99%

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

et al. 2011

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D proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the ؊10 element, and this motif is known to be recognized by region 2.4 and critical for promoter activity.In the course of efforts to define gene expression determinants from the thermophilic bacterium Aeribacillus pallidus AC6 (2, 18), we identified flagellin (Hag) as being among the most highly expressed proteins in this strain. To determine the basis for high-level Hag expression, we isolated and sequenced the hag gene and identified and expressed the protein required for its expression in The resulting peptide sequences displayed high similarity to B. subtilis flagellin (NAQDGISLIQTAEGALTETHAILQR had 96% identity with amino acids [aa] 65 to 89 and LEHTINNL GTSAENLTAAESR had 85% identity with aa 242 to 262). Two degenerate primers (FlaF1 and FlaR1) were used to amplify the flagellin gene (hag) from A. pallidus AC6 chromosomal DNA. An ϳ450-bp product was cloned into pGEM-T Easy (Promega) for DNA sequencing. The remainder of hag and its upstream region were obtained by inverse PCR. The 828-bp hag gene encodes a 275-amino-acid (29.7-kDa) protein having 63% identity with B. subtilis Hag and is preceded by a typical D promoter. To identify sigD, two degenerate primers were used to amplify a PCR fragment which was cloned into the pGEM-T Easy vector system and sequenced. The flanking portions of sigD were obtained by inverse PCR, and the gene was sequenced. The 771-bp sigD gene encodes a 256-amino-acid (28.7-kDa) protein having 67% overall identity with D Bs , with the highest levels of similarity concentrated in conserved regions 2 and 4, known to mediate promoter recognition.A. pallidus AC6 flagellin is expressed from a D -dependent promoter. Transcription of hag initiates from a canonical Ddependent promoter at a G residue 79 bp upstream of the start codon (Fig. 1). Analysis of hag::lacZ fusions integrated into B. subtilis CU1065 and HB4035 (sigD::kan) indicated that activity was D dependent and highest at late logarithmic phase, as previously reported for B. subtilis (19). Optimal promoter activity required an AT-rich region just upstream of the Ϫ35 element (Table 1), which has similarity with the upstream promoter (UP) element previously described for B. subtilis hag (6). Sequence inspection suggests that high-level Hag expression may also benefit from a strong ribosome-binding site and stabilization of the mRNA by a 5Ј hairpin sequence (24). Purification D of A. pallidus and B. subtilis and reconstitution of D RNAP. D proteins from A. pallidus AC6 and B. subtilis were expressed under T7 RNA polymerase (RNAP) control in Escherichia coli BL21(DE3)/pLysS (Novagen) by using pECG1 and pECB1 (Table 2). For purification, inclusion bodies were solubilized with Sarkosyl (1), refolded, and purified using DEAE-Sepharose and heparin-Sepharose chromatography as described previously (9). B. subtilis core RNAP was purified...

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“…Free factors generally do not specifically bind promoter DNA, and the N-terminal 1.1 region is autoinhibitory (4, 9). 1.1 might act indirectly to inhibit promoter binding by stabilizing a compact conformation of that is incompatible with promoter recognition (47). Binding to core RNAP induces large movements of the domains (3), converting into an active conformation in which the DNA binding determinants in 2 and 4 are exposed (32).…”

Section: Discussionmentioning

confidence: 99%

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

et al. 2010

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Proteins that bind factors typically attenuate the function of the factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor S (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of S -dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a ⌬crl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-S interaction and that residues 1 to 71 in S are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.In bacteria, transcription depends on a multisubunit RNA polymerase (RNAP) consisting of a catalytically active core enzyme (E) with a subunit structure ␣ 2 ␤␤Ј that associates with any one of several factors to form different holoenzyme (E) species. The subunit is required for specific promoter binding, and different factors direct RNAP to different classes of promoters, thereby modulating the gene expression patterns (17). The RNA polymerase holoenzyme containing the 70 subunit is responsible for the transcription of most genes during exponential growth (17). When cells enter stationary phase or are under specific stress conditions (high osmolarity, low pH, or high and low temperatures) during exponential growth, S , which is encoded by the rpoS gene, becomes more abundant, associates with the core enzyme, and directs the transcription of genes essential for the general stress response and for stationary phase survival (17,20,25).Sigma factors compete for binding to a limited amount of the core polymerase (16,17,20,34).70 is abundant throughout the growth cycle and has the highest affinity of all sigma factors for E in vitro (20). In contrast, levels of S reach only about one-third of the 70 levels upon entry into stationary phase, and S exhibits the lowest affinity for E of all sigma factors in vitro (20). The cell uses at least two strategies to ensure the switch between 70 -and S -associated RNA polymerases and to allow gene expression to be reprogrammed upon entry into stationary phase. Several factors (Rsd, 6S RNA, ppGpp, and D...

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Disulfide cross-linking indicates that FlgM-bound and free σ ²⁸ adopt similar conformations

Cited by 23 publications

References 23 publications

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

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Disulfide cross-linking indicates that FlgM-bound and free σ 28 adopt similar conformations

Cited by 23 publications

References 23 publications

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ D Proteins

Identification of Conserved Amino Acid Residues of the Salmonella σ S Chaperone Crl Involved in Crl-σ S Interactions

Contact Info

Product

Resources

About

Disulfide cross-linking indicates that FlgM-bound and free σ ²⁸ adopt similar conformations

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions