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At least 19 recordsLinked to original sources
PubMed1989

Escherichia coli sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar gene promoters in vitro.

A set of the periodically regulated flagellar (fla) genes of Caulobacter crescentus contain conserved promoter sequence elements at -24 and -12 that are very similar to the sequence of the nitrogen assimilation (Ntr) and nitrogen fixation (Nif) promoters of enteric bacteria and Rhizobium spp. Transcription from Ntr and Nif promoters requires RNA polymerase containing sigma 54 instead of the usual sigma 70 and, in the case of the Ntr promoters, is activated by the transcription factors NRI and NRII. We have now demonstrated that the C. crescentus flbG and flaN promoters, which contain the Ntr/Nif type of consensus sequence, are utilized by purified Escherichia coli sigma 54 RNA polymerase (E sigma 54) in the presence of NRI and NRII but not by the purified sigma 70 RNA polymerase (E sigma 70) of E. coli. Oligonucleotide-generated flbG promoter deletions that removed the highly conserved GG dinucleotide at -24 or the GC dinucleotide at -12 or altered the spacing between the -24 and -12 sequence elements prevented utilization of the flbG promoter by the E. coli E sigma 54. Transversions of T to G at positions -26 and -15 also inactivated flbG promoter function in the E. coli cell-free transcription system, while a transition of G to A at position -16 in the nonconserved spacer region had no effect. The C. crescentus flaO and flbF promoters, which do not contain the Ntr/Nif-type promoter consensus sequence, were not utilized by either purified E sigma 54 or E sigma 70 from E. coli. Our results help to define the features of the Ntr/Nif-type consensus sequence required for promoter utilization by purified E. coli E sigma 54 and support the idea that C. crescentus may contain a specialized polymerase with similar promoter specificity required for expression of a set of fla genes.

Bacteria↗
PubMed2003

Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element.

The sigma 54 promoter specificity factor is distinct from sigma 70-type factors. The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70. Here, sigma 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of sigma 54 is close to and may therefore interact with the consensus -24 promoter element. We show that the spatial relationship between the sigma 54-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between sigma 54-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some -12 promoter region-sigma 54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the beta-subunit flap domain contributes to different sets of sigma-promoter DNA interactions at sigma 54- and sigma 70-dependent promoters.

Amino Acid Sequence↗
PubMed1989

Transcriptional activation of the Klebsiella pneumoniae nifLA promoter by NTRC is face-of-the-helix dependent and the activator stabilizes the interaction of sigma 54-RNA polymerase with the promoter.

Activation of transcription at the Klebsiella pneumoniae nifLA promoter requires the phosphorylated form of the positive control protein NTRC, together with RNA polymerase modified by the alternative sigma factor sigma 54. Dimethylsulphate and potassium permanganate were used as probes to analyse the interaction of NTRC and sigma 54-RNA polymerase with supercoiled nifLA promoter DNA in vitro. In contrast to the glnAp2 promoter, sigma 54 holoenzyme did not protect guanine residues in the nifLA promoter from methylation in the absence of the activator. We propose that NTRC stabilizes the interaction of sigma 54-RNA polymerase with the -24, -12 region, in addition to its role in catalysing open complex formation. Phosphorylated NTRC binds to two sites located greater than 100 nucleotides upstream of the -24, -12 region; it also induces hyper-methylation of a G residue at -23. Enhanced methylation at -23 is not co-operative with the binding of activator to the upstream sites and may account for the ability of NTRC, when present at high concentration, to activate transcription in the absence of the upstream binding sites. The insertion of spacer mutations at -86 indicates that transcriptional activation of the nifLA promoter at low NTRC concentrations is face-of-the-helix dependent, both in vivo and in vitro. We propose that correct positioning of activator molecules at the upstream binding sites stabilizes the interaction of sigma 54-RNA polymerase with the downstream region via the formation of a DNA loop.

Bacterial Proteins↗
PubMed1997

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants.

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus -12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP beta-gamma hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Amino Acid Sequence↗
PubMed1996

ATP binding to the sigma 54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA.

The events that take place at the prokaryotic enhancer of the Pu promoter of Pseudomonas putida prior to the engagement of the sigma 54-RNA polymerase (sigma 54-RNAP) have been studied in vitro. ATP hydrolysis by XylR, the cognate regulator of the system, is preceded by the multimerization of XylR at the enhancer, which is itself triggered by the sole allosteric effect of ATP binding to the protein. Since ADP is unable to support multimerization, ATP hydrolysis might be followed by a return to the nonmultimerized state. This notion is supported further by the properties of mutant proteins that seem to be frozen, in either the nonmultimerized or the multimerized state, respectively. These results support a cyclic mechanism of ATP-dependent association/dissociation of XylR at the promoter UAS that precedes any involvement of the polymerase in transcription initiation.

Adenosine Triphosphatases↗
PubMed2000

The role of region II in the RNA polymerase sigma factor sigma(N) (sigma(54)).

Bacterial RNA polymerase holoenzymes containing the sigma subunit sigma(N) (sigma(54)) can form a stable closed complex with promoter DNA but only undergo transition to an open complex and transcription initiation when acted on by an activator protein. Proteins of the sigma(N) family have a conserved N-terminal region of 50 amino acids (Region I) that is separated from a conserved C-terminal region of around 360 amino acids (Region III) by a much more variable sequence of between 30 and 110 residues (Region II). We have investigated the role of Region II in Klebsiella pneumoniae sigma(N) by studying the properties of deletions of all or part of the region both in vivo and in vitro. We found that whilst Region II is not essential, deletion of all or part of it can significantly impair sigma(N) activity. Deletions have effects on DNA binding by the isolated sigma factor and on holoenzyme formation, but the most marked effects are on transition of the holoenzyme from the closed to the open complex in the presence of the activator protein.

Amino Acid Sequence↗
PubMed2001

Transcription of the ibpB heat-shock gene is under control of sigma(32)- and sigma(54)-promoters, a third regulon of heat-shock response.

The expression of the ibpAibpB heat-shock operon of Escherichia coli was found previously not to conform to the known pattern of expression of the sigma(32)-regulated operons because the rpoH gene mutation inactivating the sigma(32) protein did not abolish the ibp induction. We show here that this effect can depend partly on the sigma(54)-promoter that is inducible by heat shock, located upstream of the ibpB, the distal gene of the operon. It may also depend on a metabolic signal, postulated by others, and possibly required for the expression of the ibpAB genes. Thus, the ibpB gene can be translated from the transcript covering the whole operon starting from the sigma(32)-promoter and from the ibpB gene transcript starting from the sigma(54)-promoter. These results indicate that the ibpB gene is a second member of the sigma(54)-heat-shock regulon in E. coli besides pspA-E operon. Thus, heat-shock response involves three regulons controlled by sigma(32), sigma(24), and sigma(54) RNA polymerase subunits.

Bacterial Proteins↗
PubMed1996

The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA.

An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase. It requires the presence of the upstream activation sequence on supercoiled DNA. Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM. Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place. The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration. On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate. These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA. In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.

Base Sequence↗
PubMedDate not supplied

The role of FlbD in regulation of flagellar gene transcription in Caulobacter crescentus.

The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD. FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by sigma 54 RNA polymerase (E sigma 54) and its function is required for transcription of the class III and IV fla genes. Here we show that purified FlbD binds to ftr elements that are required for transcription from the sigma 54-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli E sigma 54, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site. Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbDS140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrCS160F), displayed a greater potential for activating E sigma 54-dependent transcription that the wildtype protein. We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD. Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by E sigma 54 and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.

Bacterial Proteins↗
PubMed1993

In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N).

Bacteria synthesize a number of different sigma factors which allow the co-ordinate expression of groups of genes owing to the ability of sigma to confer promoter-specific transcription initiation on RNA polymerase. In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, sigma 70. A clear exception is the sigma factor sigma 54 (sigma N), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes. Studies of sigma 54 (sigma N) have demonstrated that this sigma is quite distinct both structurally and functionally from the sigma 70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with sigma 70 and its relatives.

Amino Acid Sequence↗
PubMed2004

Role of the amino-terminal GAF domain of the NifA activator in controlling the response to the antiactivator protein NifL.

The NifA protein from Azotobacter vinelandii belongs to a family of enhancer binding proteins (EBPs) that activate transcription by RNA polymerase containing the sigma factor sigma(54). These proteins have conserved AAA+ domains that catalyse ATP hydrolysis to drive conformational changes necessary for open complex formation by sigma(54)-RNA polymerase. The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino-terminal GAF domain of NifA that binds 2-oxoglutarate. Mutations conferring resistance to NifL are located in both the GAF and the AAA+ domains of NifA. To investigate the mechanism by which the GAF domain regulates the activity of the AAA+ domain, we screened for second-site mutations that suppress the NifL-resistant phenotype of mutations in the AAA+ domain. One suppressor mutation, F119S, in the GAF domain restores inhibition by NifL to an AAA+ domain mutation, E356K, in response to fixed nitrogen but not in response to oxygen. The biochemical properties of this mutant protein are consistent with the in vivo phenotype and demonstrate that interdomain suppression results in sensitivity to inhibition by NifL in the presence of the signal transduction protein GlnK, but not to the oxidized form of NifL. In the absence of an AAA+ domain mutation, the F119S mutation confers hypersensitivity to repression by NifL. Isothermal titration calorimetry demonstrates that this mutation prevents binding of 2-oxoglutarate to the GAF domain. Our data support a model in which the GAF domain plays an essential role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. These observations are of general significance in considering how the activities of EBPs are controlled in response to environmental signals.

Adenosine Triphosphatases↗
PubMed1994

Molecular analysis of the operon which encodes the RNA polymerase sigma factor sigma 54 of Escherichia coli.

The rpoN gene (encoding the sigma factor sigma 54) of Escherichia coli was cloned and its nucleotide sequence determined. Promoter probe analysis confirmed the presence of a promoter in a 350 bp fragment covering the start of rpoN. The likely promoter was identified. The nucleotide sequence of the region extending 2.1 kb downstream of rpoN was also determined. This region contained four open reading frames encoding potential polypeptides of 10750, 17959, 32492 and 9810 Da; maxicell and T7 promoter studies showed that four polypeptides of similar molecular masses were expressed from this region. The amino acid sequence of the 17959 Da polypeptide showed homology to the enzyme IIA domains of several proteins of the bacterial sugar phosphotransferase system (PTS), and the 9810 Da polypeptide showed homology to the HPr proteins of the bacterial PTS. The proteins encoded downstream of rpoN are known to negatively regulate sigma 54 activity. The homologies therefore suggest that this effect on sigma 54 may be mediated by sequential protein phosphorylation and suggest that there is a link between signal transduction and transcription of sigma 54-dependent genes.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Amino Acid Sequence↗
PubMed2003

The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54.

Members of the protein family called ATPases associated with various cellular activities (AAA(+)) play a crucial role in transforming chemical energy into biological events. AAA(+) proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA(+) mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the sigma(54)-RNA polymerase. Closed promoter complexes formed by the sigma(54)-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one sigma(54)-binding surface in the PspF AAA(+) domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one sigma(54)-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between sigma(54) and PspF are transient and could be part of a mechanism used generally by other AAA(+) proteins to regulate activity.

Adenosine Triphosphatases↗
PubMed1997

Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy.

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.

Bacterial Proteins↗
PubMed1989

In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex.

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25. The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.

Bacterial Proteins↗
PubMed1991

Cassette mutagenesis implicates a helix-turn-helix motif in promoter recognition by the novel RNA polymerase sigma factor sigma 54.

Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.

Amino Acid Sequence↗
PubMed1993

Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators.

The catabolic plasmid pVI150 of Pseudomonas sp. strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives. The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence. Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN). Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter. Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR. The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter. Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters. The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively. The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators. This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.

Amino Acid Sequence↗