255 results on '"Dixon, Ray"'
Search Results
252. A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii.
- Author
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Martinez-Argudo I, Little R, and Dixon R
- Subjects
- Adenosine Diphosphate metabolism, Amino Acid Substitution, Arginine chemistry, Azotobacter vinelandii genetics, Bacterial Proteins genetics, Base Sequence, DNA, Bacterial genetics, Genes, Bacterial, Ketoglutaric Acids pharmacology, Models, Biological, Mutagenesis, Site-Directed, Nitrogen Fixation genetics, Protein Conformation, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, Azotobacter vinelandii metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Nitrogen Fixation physiology
- Abstract
NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of sigma(54)-dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of protein-protein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.
- Published
- 2004
- Full Text
- View/download PDF
253. In silico analysis of the sigma54-dependent enhancer-binding proteins in Pirellula species strain 1.
- Author
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Studholme DJ and Dixon R
- Subjects
- Bacteria genetics, Bacterial Proteins genetics, Base Sequence, DNA, Bacterial analysis, DNA-Directed RNA Polymerases genetics, Enhancer Elements, Genetic genetics, Escherichia coli Proteins, Molecular Sequence Data, Phylogeny, Promoter Regions, Genetic, RNA Polymerase Sigma 54, Regulon genetics, Sigma Factor genetics, Bacteria metabolism, Bacterial Proteins metabolism, DNA-Binding Proteins, DNA-Directed RNA Polymerases metabolism, Enhancer Elements, Genetic physiology, Gene Expression Regulation, Bacterial, Regulon physiology, Sigma Factor metabolism
- Abstract
The planctomycetes are a phylogenetically distinct group of bacteria, widespread in aquatic and terrestrial environments. Their cell walls lack peptidoglycan and their compartmentalised cells undergo a yeast-like budding cell division process. Many bacteria regulate a subset of their genes by an enhancer-dependent mechanism involving the alternative sigma factor sigma54 (RpoN, sigmaN) in association with sigma54-dependent transcriptional activators known as enhancer-binding proteins (EBPs). The sigma54-dependent regulon has previously been studied in several groups of bacteria, but not in the planctomycetes. We wished to exploit the recently published complete genome sequence of Pirellula species strain 1 to predict and analyse the sigma54-dependent regulon in this interesting group of bacteria. The genome of Pirellula species strain 1 encodes one homologue of sigma54, and 16 sigma54-dependent EBPs, including 10 two-component response regulators and a homologue of Escherichia coli RtcR. Two EBPs contain forkhead-associated domains, representing a novel protein domain combination not previously observed in bacterial EBPs and suggesting a novel link between the enhancer-dependent regulon and 'eukaryotic-like' protein phosphorylation in bacterial signal transduction. We identified several potential sigma54-dependent promoters upstream of genes and operons including two homologues of csrA, which encodes the global regulator CsrA, and rtcBA, encoding a RNA 3'-terminal phosphate cyclase. Phylogenetic analysis of EBP sequences from a wide range of bacterial taxa suggested that planctomycete EBPs fall into several distinct clades. Also the phylogeny of the sigma54 factors is broadly consistent with that of the host organisms. These results are consistent with a very ancient origin of sigma54 within the bacterial lineage. The repertoire of functions predicted to be under the control of the sigma54-dependent regulon in Pirellula shares some similarities (e.g. rtcBA) as well as exhibiting differences with that in other taxonomic groups of bacteria, reinforcing the evolutionarily dynamic nature of this regulon.
- Published
- 2004
- Full Text
- View/download PDF
254. Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae.
- Author
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Ray P, Smith KJ, Parslow RA, Dixon R, and Hyde EI
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, Binding Sites genetics, DNA genetics, DNA Fingerprinting, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Magnetic Resonance Spectroscopy, Oligonucleotides genetics, Oligonucleotides metabolism, Protein Binding, Protein Structure, Secondary, Transcription Factors chemistry, Transcription Factors genetics, Bacterial Proteins metabolism, DNA metabolism, Klebsiella pneumoniae metabolism, Transcription Factors metabolism
- Abstract
The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen fixation (nif) operons except the regulatory nifLA genes. At these operons, NifA binds to an upstream activator sequence (UAS), with the consensus TGT-N(10)-ACA, via a C-terminal DNA-binding domain (CTD). Binding of the activator to this upstream enhancer-like sequence allows NifA to interact with RNA polymerase containing the alternative sigma factor, sigma(54). The isolated NifA CTD is monomeric and binds specifically to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experiments have been used to assign the signals from the protein backbone. Three alpha-helices have been identified, based on secondary chemical shifts and medium range Halpha(i)-NH(i)( + 1), and NH(i)-NH(i)( + 1) NOEs. On addition of DNA containing a half-site UAS, several changes are observed in the NMR spectra, allowing the identification of residues that are most likely to interact with DNA. These occur in the final two helices of the protein, directly confirming that DNA binding is mediated by a helix-turn-helix motif.
- Published
- 2002
- Full Text
- View/download PDF
255. Direct interaction of the NifL regulatory protein with the GlnK signal transducer enables the Azotobacter vinelandii NifL-NifA regulatory system to respond to conditions replete for nitrogen.
- Author
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Little R, Colombo V, Leech A, and Dixon R
- Subjects
- Calorimetry, Ketoglutaric Acids metabolism, Kinetics, Peptide Fragments metabolism, Plasmids, Recombinant Proteins metabolism, Signal Transduction, Trypsin, Azotobacter vinelandii metabolism, Bacterial Proteins metabolism, Carrier Proteins metabolism, Nitrogen Fixation
- Abstract
The Azotobacter vinelandii NifL-NifA regulatory system integrates metabolic signals for redox, energy, and nitrogen status to fine tune regulation of the synthesis of molybdenum nitrogenase. The NifL protein utilizes discrete mechanisms to perceive these signals leading to the formation of a protein-protein complex, which inhibits NifA activity. Whereas redox signaling is mediated via a flavin-containing PAS domain in the N-terminal region of NifL, the nitrogen status is sensed via interaction with PII-like signal transduction proteins and small molecular weight effectors. The nonuridylylated form of the PII-like protein encoded by A. vinelandii glnK (Av GlnK) stimulates NifL to inhibit transcriptional activation by NifA in vitro. Here we demonstrate that the nonmodified form of Av GlnK directly interacts with A. vinelandii NifL and that this interaction is dependent on Mg(2+), ATP, and 2-oxoglutarate. Differences were observed in the regulation of the Av GlnK-NifL interaction by 2-oxoglutarate compared with the role of this effector in modulating the interaction of enteric PII-like proteins with their receptors. We also report that the interaction between Av GlnK and NifL is abolished by site-directed substitution of a single amino acid in the T-loop region of Av GlnK and that uridylylation of the conserved tyrosine residue in the T-loop inhibits the interaction. No association was detected between Av GlnK and the N-terminal region of NifL and our results demonstrate that Av GlnK directly interacts with the C-terminal histidine protein kinase-like domain.
- Published
- 2002
- Full Text
- View/download PDF
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