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

Monteil, Véronique; Kolb, Annie; d’Alayer, Jacques; Béguin, Pierre; Norel, Françoise

doi:10.1128/jb.01197-09

Cited by 16 publications

(55 citation statements)

References 54 publications

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“…Surface-exposed conserved residues identified from our BTH analyses are shown in teal, as are F53 and Y22, which were identified in a previous study (48). Residue E52 (labeled "hidden") is surface exposed but is not visible in this view.…”

Section: Structure Of P Mirabilis Crlmentioning

confidence: 99%

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Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

et al. 2014

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bBacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the S regulon by binding to S to promote its association with core RNAP. We recently characterized the determinants in S responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-S interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with S . The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to S . Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of E S -dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the S -Crl interaction.

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Section: Structure Of P Mirabilis Crlmentioning

confidence: 99%

“…100% conserved were replaced with alanines (48). Four of the substitutions in that screen met our analysis criteria, namely, Y22A, R51A, E52A, and F53A.…”

Section: Structure Of P Mirabilis Crlmentioning

confidence: 99%

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

et al. 2014

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“…In this system, the T25-S and Crl-T18 hybrid proteins were shown to interact, yielding levels of ␤-galactosidase activity higher than those detected in negative controls (Fig. 4B) (28). We previously showed that the first 71 residues of S were not required for Crl binding since the T25- and Crl-T18 chimeras interacted efficiently (28 and Fig.…”

Section: G282 Is Located In a Flexible Loop In Region 4 Ofmentioning

confidence: 99%

“…Several factors (Rsd, 6S RNA, ppGpp, and DksA) indirectly increase S competitiveness by decreasing the ability of 70 to bind to E or by inhibiting E 70 activity (21). Furthermore, the nonconventional regulatory protein Crl increases the performance of S (14,28,35,37,39,40,46). Unlike classical regulators of transcription, Crl binds S instead of DNA (3,12), and the transient interaction of Crl with S increases the association rate of S to E (12), thereby facilitating RNAP holoenzyme E S formation (12,14,46).…”

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confidence: 99%

“…The resulting cyclic AMP binds to and activates the transcription activator CRP, a positive regulator of the lac and mal operons involved in lactose and maltose catabolism. Derivatives of pUT18 and pKT25, used in the BACTH assays, were constructed by cloning PCR-amplified DNA fragments encoding the protein of interest from S. Typhimurium between the PstI and EcoRI sites of pUT18 and the XbaI and KpnI sites of pKT25 as previously described (Table 1) (28). All plasmids were confirmed to be correct by DNA sequencing.…”

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Crl Binds to Domain 2 of σ ^S and Confers a Competitive Advantage on a Natural rpoS Mutant of Salmonella enterica Serovar Typhi

et al. 2010

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The RpoS sigma factor ( S ) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS Ty19 ) led to the synthesis of a S Ty19 protein carrying a single glycine-to-valine substitution at position 282 in S domain 4, which was much more dependent than the wild-type S protein on activation by Crl, a chaperone-like protein that increases the affinity of S for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of S domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of S Ty19 to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the E S holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between S and E. The rpoS Ty19 allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in ⌬crl populations. Thus, these results indicate that the competitive advantage of the rpoS Ty19 mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.In bacteria, transcription depends on a 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 E holoenzymes. 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 holoenzyme containing the 70 subunit is responsible for the transcription of most genes during exponential growth (17). When cells enter stationary phase or undergo 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 E, and directs the transcription of genes essential for the general stress response (17,18,21). In Salmonella and Escherichia coli, the S regulon comprises more than 300 genes contributing to survival during stationary phase, adaptive stress responses, biofilm formation, and virulence of S. enterica serovar Typhimurium, a wide-host-range pathogen and a major cause of human gastroenteritis and food-borne disease (2, 48). However, the precise function of nearly half of the genes in the regulon is unknown.An intriguing aspect of S is the allelic variation of rpoS in E.coli and Salmonella (9,13,36,38,50). Because of its role in general stress resistance and its high position in the hierarchy of transcriptional regulators, one might expect S to be conser...

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1H, 13C and 15N resonance assignments of σS activating protein Crl from Salmonella enterica serovar Typhimurium

2015

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The general stress response in Enterobacteria, like Escherichia coli or Salmonella, is controlled by the transcription factor σ(S), encoded by the rpoS gene, which accumulates during stationary phase growth and associates with the core RNA polymerase enzyme (E) to promote transcription of genes involved in cell survival. Tight regulation of σ(S) is essential to preserve the balance between self-preservation under stress conditions and nutritional competence in the absence of stress. Whereas σ factors are generally inactivated upon interaction with anti-sigma proteins, σ(S) binding by the Crl protein facilitates the formation of the holoenzyme Eσ(S), and therefore σ(S)-controlled transcription. Previously, critical residues in both Crl and σ(S) were identified and assigned to the binding interface in the Crl-σ(S) complex. However, high-resolution structural data are missing to fully understand the molecular mechanisms underlying σ(S) activation by Crl, in particular the possible role of Crl in triggering domain rearrangements in the multi-domain protein σ(S). Here we provide the (1)H, (13)C and (15)N resonance assignments of Salmonella enterica serovar Typhimurium Crl, as a starting point for CrlSTM structure determination and further structural investigation of the CrlSTM-σ STM (S) complex.

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

Cited by 16 publications

References 54 publications

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Crl Binds to Domain 2 of σ ^S and Confers a Competitive Advantage on a Natural rpoS Mutant of Salmonella enterica Serovar Typhi

1H, 13C and 15N resonance assignments of σS activating protein Crl from Salmonella enterica serovar Typhimurium

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

Cited by 16 publications

References 54 publications

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Crl Binds to Domain 2 of σ S and Confers a Competitive Advantage on a Natural rpoS Mutant of Salmonella enterica Serovar Typhi

1H, 13C and 15N resonance assignments of σS activating protein Crl from Salmonella enterica serovar Typhimurium

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

Crl Binds to Domain 2 of σ ^S and Confers a Competitive Advantage on a Natural rpoS Mutant of Salmonella enterica Serovar Typhi