The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of <i>Klebsiella pneumoniae</i>

Drummond, Martin; Contreras, Asunción; Mitchenall, Lesley A.

doi:10.1111/j.1365-2958.1990.tb02012.x

Cited by 108 publications

(104 citation statements)

References 32 publications

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“…Table 1 for a summary of properties). These results confirm the modular nature of the central catalytic domain of the NIFA protein from K. pneumoniae in that this domain itself and all four fusion proteins containing it (the other two being MBP-NIFA and MBP-central domain) are active in vivo (10,25) and in vitro. This is in contrast to the case for NTRC, a well-studied homolog of NIFA: neither MBP-⌬N-NTRC nor MBP-NTRC-⌬C is active, and the same is true for ⌬N-NTRC and NTRC-⌬C (28,40).…”

Section: P]gtp or [␥-supporting

confidence: 70%

In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA

et al. 1995

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We previously showed that the same was true for a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active. Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out. Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K. pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo. This was true independent of the activator, i.e., with MBP-NIFA and MBP-⌬N-NIFA and with the homologous activator NTRC. When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was favored and the complexes formed were distinguishable from those formed in the presence of other nucleotides, probably as a consequence of initiation of transcription.The NIFA protein is the central positive regulator of expression of the nitrogen fixation (nif) genes in diazotrophic species of purple bacteria (proteobacteria) (13). NIFA activates transcription of nif genes by the alternative holoenzyme form of RNA polymerase containing the 54 factor (E ⅐ 54

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Section: P]gtp or [␥-supporting

confidence: 70%

In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA

et al. 1995

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“…mechanism, deletions that truncate or remove the receiver domain result in constitutively active proteins (see Doucleff et al 2005). In stark contrast, positively regulated activators like NtrC require the presence of the phosphorylated receiver domain to function in ATPase and transcription activation assays (Drummond et al 1990;Weiss et al 1992a,b;Klose et al 1993). Our structures, together with previous biochemical data showing that the activated receiver domain of one subunit is in contact with the ATPase domain of a second, nonidentical subunit, offer an explanation for the positive mode of regulation in NtrC.…”

Section: Mechanism Of Positive Activation In 54 -Dependent Aaa+ Atpasmentioning

confidence: 99%

The structural basis for regulated assembly and function of the transcriptional activator NtrC

Carlo¹,

Chen²,

Hoover³

et al. 2006

Genes Dev.

111

180

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In two-component signal transduction, an input triggers phosphorylation of receiver domains that regulate the status of output modules. One such module is the AAA+ ATPase domain in bacterial enhancer-binding proteins that remodel the 54 form of RNA polymerase. We report X-ray solution scattering and electron microscopy structures of the activated, full-length nitrogen-regulatory protein C (NtrC) showing a novel mechanism for regulation of AAA+ ATPase assembly via the juxtaposition of the receiver domains and ATPase ring. Accompanying the hydrolysis cycle that is required for transcriptional activation, we observed major order-disorder changes in the GAFTGA loops involved in 54 binding, as well as in the DNA-binding domains. The 54 holoenzyme form of RNA polymerase forms closed DNA complexes that open only with the help of enhancer-binding proteins (EBPs). For the nitrogen-regulatory protein C of enteric bacteria (NtrC), such activity starts a cascade of events that may ultimately lead to the activation of transcription for as much as 2% of the genome (Zimmer et al. 2000). Sequence analysis of >160054 -dependent transcriptional activators shows that in addition to their oligomerization/AAA+ ATPase domain, they also have a DNA-binding domain and a regulatory domain, which in 50% of cases is a twocomponent receiver domain (Bateman et al. 2004). The helix-turn-helix DNA-binding domain recognizes enhancer-like sequences between 100 and 150 bp upstream of the promoter. In their inactive states these proteins are usually dimers that bind to pairs of tandem sites in the enhancer elements. Upon activation via the regulatory domains, they oligomerize into ATPase-active rings that use the energy from ATP hydrolysis to physically remodel closed complexes of 54 holoenzyme and promoter DNA (Rombel et al. 1998;Neuwald et al. 1999;Chaney et al. 2001;Lee et al. 2003).Crystal or NMR structures are available for several isolated domains and truncation constructs of EBPs including NtrC, DctD, PspF, ZraR, and NtrC1 (Volkman et al. 1995;Kern et al. 1999;Pelton et al. 1999;Meyer et al. 2001;Park et al. 2002;Hastings et al. 2003;Lee et al. 2003;Doucleff et al. 2005;Rappas et al. 2005;Sallai and Tucker 2005). Existing structural information on DctD and NtrC1 has been used to provide a model of how two-component signal transduction can regulate assembly of their AAA+ ATPase domains ), but this model fails to explain regulation in the closely related protein NtrC (40% sequence identity, 60% sequence similarity) Hastings et al. 2003). Here we report both SAXS/WAXS (small-and wide-angle X-ray scattering) and EM (electron microscopy) structures of the full-length, activated form of NtrC from Salmonella typhimurium. Docking of atomic models for the individual domains into the structures reveals their organization within the activated ring and uncovers a novel mechanism for the use of two-compo-

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“…The cells wcre then spun down, resuspended in 50% glycerol/saline phosphate, and stored at -20°C. Small aliquots were plated on Luria-Bertani agar [6] genesis, using template purified by isopycnic centrifugation, as previously described [7]. When degenerate oligonucleotides were employed.…”

Section: Plasmid Construction and Mutagenesismentioning

confidence: 99%

Roulette mutagenesis of the FMN‐binding site of Klebsiella pneumoniae flavodoxin

Drummond

Huff

Green

1993

European Journal of Biochemistry

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A method of randomising specific regions of coding sequences has been devised which utilises the Lac phenotype to identify mutants. Intact genes can be mutagenised, making it unnecessary to reclone the mutations before examining mutant phenotypes. The method has been applied to three residues around the N-terminus of the first a helix of the Klebsiella pneumoniue nitrogenase flavodoxin, which are predicted to form part of the phosphate-binding subsite. Surprisingly, most substitutions at Gly12, a highly conserved residue in the chain reversal preceding the a helix, appeared to be fairly stable in vivo and were found to retain some function. Substitutions at Lysl3, a surface residue which contributes to a patch of positive charge characteristic of the nitrogenase flavodoxins, had no major effect on stability or function. However, most substitutions at Thrl4, which is prcdicted to hydrogen bond to the phosphate of the prosthetic group FMN, were much more destabilising and grossly reduced function. The exceptions were Ala, Cys, Ser and Val, which suggests that the bulk of the residue at this position is critical.The power of oligonucleotide-directed mutagenesis in the analysis of protein structure and function has been greatly enhanced by the introduction of techniques which employ oligonucleotides containing randomised segments. Such degenerate oligonucleotides allow a range of substitutions to be isolated in one experiment, which in genetic analysis provides some safeguard against overinterpreting the effects of single mutations, and yields a range of mutant proteins from which the most interesting can be selected for biochemical and biophysical analysis. We have developed a powerful new method using degenerate oligonucleotides which we call 'roulette mutagenesis', and applicd it to the flavodoxin encoded by nifF in Klebsiella pneurnoniue.This flavodoxin is the immediate electron donor to component 2 of nitrogenase. It shows strong sequence similarity to other flavodoxins, which have a common structure comprising a parallel / 3 sheet sandwiched between a hclices (for review, see [ 2 ] ) . The crystal structure of the Synechococcus PCC 6301 (Aspergillus nidulans) flavodoxin has been used to roughly model the Klebsielln molecule [l]. Tertiary structures have also been determined crystallographically for flavodoxins from Clostridiurn beijerinckii MP, Desulfovibrio vulgaris and Chondrus crispus (see [2] and references therein) and by two-dimensional 'H-NMR Tor the Mega;,-phaera elsdenii flavodoxin [31. The crystal structure of the Azotobacter chrnococcum flavodoxin, an electron donor to nitrogenase showing strong sequence similarity to the K. pneunzoniae moleculc, has recently been completed [4]. We have investigated the contribution to structure and function of three residues in the K. pneurnoniae flavodoxin, Gly12, Lysl3 and Thrl4, which form part of the binding cleft for the prosthetic group, FMN, and are close in the tertiary structure to Cys68, which we have recently shown to be covalently but reversibly modifie...

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The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae

Cited by 108 publications

References 32 publications

In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA

In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA

The structural basis for regulated assembly and function of the transcriptional activator NtrC

Roulette mutagenesis of the FMN‐binding site of Klebsiella pneumoniae flavodoxin

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