Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells. Transcription of fhuA was independent of a functional copy of rpol, a neighbouring gene that specifies a putative ECF sigma factor of RNA polymerase and which is involved in siderophore production in Rhizobium. Mutations in fhuA did not detectably affect symbiotic N2 fixation on peas. An fhuA::gus fusion was expressed by bacteria in the meristematic zone of pea nodules but not in mature bacteroids. Some other strains of R. leguminosarum also contain a pseudogene version of fhuA. The sequences of some of these and the 'real' fhuA genes were determined.
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PMID:Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains. 1078 41

Two distinct classes of RNA polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38). This indicates a common use of sets of sequences in core for interacting with the two sigma classes. Some sites chosen in sigma(54) for modification with Fe-BABE were positions, which when mutated, deregulate the sigma(54)-holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in sigma(54) may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of sigma(54) proximity to the core is apparently not evident with sigma(70), and may represent a specialized interaction.
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PMID:Conservation of sigma-core RNA polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes. 1085 47

We are interested in learning how iron is safely inserted and stored in ferritin. Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L). In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem. Biophys. Res. Commun. 242, 39-45 (1998)). This results in the protein being expressed at very low levels. This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG). Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit. Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios. Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition.
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PMID:Production of recombinant human apoferritin heteromers. 1114 22

The overlapping and opposing promoter elements for the Escherichia coli fepDGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their -10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the E. coli consensus sequence, both promoters were poorly conserved at the -35 position and mutations which strengthened the -35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the fepD and ybdA primary promoters were shown to require a 5'-TGn-3' upstream extension of their -10 elements for optimal activities. Secondary promoters were identified for both fepD and ybdA, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping fepD and ybdA promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.
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PMID:Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli. 1122 6

The alternative sigma factor PvdS is required by Pseudomonas aeruginosa for initiation of transcription from pyoverdine (pvd) promoters. Two divergent PvdS-dependent promoters (pvdE and pvdF) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other pvd promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from pvd promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from pvd promoters and are likely to represent a -35 sequence element for this sigma factor.
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PMID:Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa. 1122 21

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.
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PMID:Regulatory sequences in sigma 54 localise near the start of DNA melting. 1124 80

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia. The anaerobic incorporation of oxygen-sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding. Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen-sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway. The C122 variant failed to assemble iron-sulphur clusters and to bind DNA. Second-site substitutions that partially restore activity to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was concluded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.
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PMID:Anaerobic acquisition of [4FE 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions. 1125 37

Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit. The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors.
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PMID:Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase. 1129 18

The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
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PMID:Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine. 1136 35

Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In Paracoccus pantotrophus, the enzyme system that catalyzes this reaction is encoded by the narKGHJI gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from narK is narR, a gene encoding a member of the FNR family of transcriptional activators. narR is transcribed divergently from the other nar genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and narK but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the sigma(70) subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription. narR is also found in Paracoccus denitrificans, indicating that this species contains at least three FNR homologues.
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PMID:Maximal expression of membrane-bound nitrate reductase in Paracoccus is induced by nitrate via a third FNR-like regulator named NarR. 1137 24


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