Martin Buck

In vivo studies on the interaction of RNA polymerase-σ54 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.

Transcriptional activation of the Klebsiella pneumoniae nitrogenase promoter may involve DNA loop formation.

Transcriptional activation of nitrogen fixation genes by Nrt A in Klebsiella pneumoniae requires an upstream Nif A binding site. We now report that the introduction of haif turns of the DNA helix into the DNA separating the upstream NifA binding site from the downstream promoter element of the nifH promoter decreases Nif A‐mediated activation to a greater extent than does the introduction of full helical turns. Reducing the spacing between the upstream and downstream elements of the nifH promoter also results in a promoter down phenotype. Introduction of a tight protein‐binding site, the lac operator, between the upstream and downstream promoter elements did not render activation of the nifH promoter sensitive to occupancy of this site by the lac repressor. These findings indicate that NifA‐mediated activation of transcription requires that NifA is bound upstream, and to the correct face of the DNA helix, in order to interact with downstream transcription factors. This implies that the interaction is brought about by the formation of a DNA loop between upstream and downstream promoter elements rather than by NifA sliding downstream.

Core RNA polymerase assists binding of the transcription factor σ;54 to promoter DNA

The Sigma subunit of bacterial RNA polymerase Is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing σ;54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The σ;54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of σ;54 to promoter DNA, An altered form of σ;54 with a deletion within the amino‐terminal region showed increased affinity for specific DNA‐binding sites. Although able to complex with core RNA polymerase the mutant σ;54 failed to respond to core polymerase in the manner characteristic of the wild‐type σ;54 by altering its footprint. This result indicates that σ;54 has a latent DNA‐binding activity which is revealed by core RNA polymerase, and possibly involves a change in σ;54 conformation. Promoter complexes which formed with σ;54‐holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.

Activator-independent formation of a closed complex between σ54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae

The alterNatlve sigma factor σ54 is required for transcription of nitrogen fixation genes in Klebsiella pneutnoniae and other diazotrophs. The nif genes, and other Eσ54‐dependent genes whose products are necessary for a wide range of processes, are positively regulated. A unifying model that is well supported by studies on nif and other nitrogen‐regulated (ntr) genes includes the central tenet that σ54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator‐independent complex between Eσ54 and the NifA‐dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the Eσ54 recognition sequence.

Central domain of the positive control protein NifA and its role in transcriptional activation.

The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.

The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA

The influence of the Klebsiella pneumoniae nifL gene product upon the interaction of the transcriptional activator protein NifA with the nifH promoter has been examined using in vivo dimethylsulphate ‘footprinting’. Binding of NifA to the upstream activator sequence (UAS) of the nifH promoter in the presence of the NifL protein was observed under nitrogen‐limiting growth conditions. Growth in the presence of NH+4 or addition of NH+4 to nitrogen‐limited cells diminished the interaction of NifA with the UAS when NifL was present. Repression of nif transcription by NifL may therefore involve an interaction between NifL and NifA which reduces the affinity of NifA for the UAS.

The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the Nif A promoter

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed σ;54‐dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60‐200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase‐σ54 complex (Eσ54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between Eσ54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase‐σ54‐binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp.

Positional requirements for the function of nif-specific upstream activator sequences.

SummaryThe upstream activator sequence (UAS) found in Klebsiella pneumoniae nif promoters and required for the activation of transcription by nifA, is absent from the nifF-nifL intergenic region, but is present downstream from the nifLA transcription start at+59. To determine whether nif upstream activator sequences can function in a 3′ position, the nifH UAS was cloned downstream from the NifH transcription start, but no activation of transcription by nifA dependent upon the UAS in its 3′ location could be detected. A mild repressive effect was detectable when the nifH UAS was placed downstream of the nifH promoter, but not when the cat promoter was substituted for the nifLA promoter upstream from the motif at+59 described above. However, deletion analysis showed that the UAS motif located downstream of the nifLA promoter has a role in transcription from the nifF promoter, although it is situated at position-263 with respect to the nifF transcription start, about 100 bp further upstream than previously described occurrences of the activator sequence.

PURIFICATION AND ACTIVITIES OF THE RHODOBACTER CAPSULATUS RPON (SIGMA N) PROTEIN

The rpoN‐encoded sigma factors (σN) are a distinct class of bacterial sigma factors, with no obvious homology to the major σ70 class. The σN‐containing RNA polymerase holoenzyme functions in enhancer‐dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatusσN protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coliσN holoenzyme, and may represent a minor subclass of σN proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatusσN protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins. As first described for the Kleb‐siella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatusσN protein and its response to core RNA polymerase was clearly different from that determined for the K. pneumoniae and E. coli proteins. Results are discussed in the context of a requirement to modulate the activity of the DNA‐binding surfaces of σN to regulate σN function. Circular dichroism was used to evaluate the structure of the R. capsulatus protein and revealed differences in the tertiary signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA‐binding domain of σN

Frameshifts close to the Klebsiella pneumoniae nifH promoter prevent multicopy inhibition by hybrid nifH plasmids

SummaryCertain multicopy plasmids bearing promoter sequences of nitrogen fixation (nif) genes inhibit expression of chromosomal genes in Nif+Klebsiella pneumoniae, hence leading to a Nif- phenotype. This ‘multicopy inhibition’ has been attributed to the titration of the nif-specific activator protein NifA by the plasmid-borne promoter sequences. We now report that multicopy inhibition by nifH translational fusions is sensitive to frameshifts close to the nifH promoter. Transcriptional nifH fusion plasmids in which translation terminated near the nifH promoter were transcriptionally active and showed multicopy inhibition; introduction of a transcription terminator after the nifH coding sequence in these plasmids prevented their multicopy inhibition. Therefore it seems likely that premature termination of transcription prevents multicopy inhibition by the nifH promoter.