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)

The coenzyme A-glutathione mixed disulfide (CoASSG), when complexed with iron, is capable of inhibiting the RNA polymerase of Escherichia coli. A modified procedure involving a short time of exposure to high salt allowed the reliable preparation of CoASSG-Fe which was active in inhibiting RNA polymerase. The CoASSG-Fe complex acted as a noncompetitive inhibitor for the incorporation of all four nucleoside triphosphates but had a greater effect on GMP and CMP incorporation than AMP and UMP incorporation. Neither temperature nor ionic-strength changes affected CoASSG-Fe inhibition, and the use of rifampicin showed that CoASSG-Fe did not inhibit either the initiation or elongation processes of the polymerase. CoASSG-Fe was a more effective inhibitor at low DNA-template concentrations and it was more effective in inhibiting the incorporation of CMP and GMP on simple dG-dC containing templates and the asymmetric polymer poly d(T-C) . poly d(G-A). The inhibition of transcription of poly d(I-C) was less effective than the inhibition of transcription of poly d(G-C). Equilibrium dialysis in microdialysis cells showed that CoASSG-Fe could associate with DNA in the absence of RNA polymerase.
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PMID:Partial characterization of the mode of inhibition of Escherichia coli RNA polymerase by the mixed disulfide, CoASSG. 37 69

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.
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PMID:Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli. 130 92

The aerobactin operon of the virulence plasmid pColV-K30 of Escherichia coli K12 consists of four genes for biosynthesis and one for transport of the siderophore. Regulation by iron occurs at the transcriptional level and is mediated by a ferrous iron binding protein designated Fur (ferric uptake regulation). The metallated Fur repressor binds at a palindromic dyad, the "iron box" operator, situated in the vicinity of the RNA polymerase attachment site of the promoter. Evidence suggests that the ferrous iron enters the C-terminal domain of Fur to cause a conformational change in the N-terminal part of the protein. This results in greatly enhanced affinity of the repressor for the operator.
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PMID:Mechanism and regulation of synthesis of aerobactin in Escherichia coli K12 (pColV-K30). 139 37

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.
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PMID:Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin. 140 58

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.
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PMID:Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli. 150 Jan 90

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.
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PMID:Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli. 164 78

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.
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PMID:Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. 184 79

This review has considered what is known about the precise chemical mechanisms involved in the signal transduction of heavy metal ions. By reviewing what is known about general modes of signal transduction, we may draw parallels with the detection of and response to metal ions. In all forms of signal transduction, sensors and transducers are required. Yet, it is apparent that each system has unique features which undoubtedly are critical for the specific signal at hand. Within the context of metal-responsive systems, regardless of whether or not the metal ion is being sequestered, directly utilized, removed or otherwise, several examples of specific metalloregulatory proteins have been elucidated and are summarized in Table II. A close inspection of Table II reveals that in most signal transduction pathways for heavy metals, the presence of the metal ion causes a marked change in the nucleic acid binding capacity of the metalloregulatory protein. For example, the presence of iron results in the dissociation of a protein from iron responsive elements, thereby derepressing ferritin translation. In other instances, metal binding allows a metalloregulatory protein to associate with DNA to activate or repress transcription, as with ACE1 and Fur, respectively. In fact, to the authors' knowledge, it appears that all characterized ligand-responsive transcription factors change nucleic acid binding activity upon ligand binding. This change in affinity is a major feature of the mechanism for activation or repression by these receptors. In contrast, the mercuric ion metalloregulatory protein, MerR, operates by an entirely different transduction mechanism. MerR remains bound to its operator sequence in the presence and absence of mercuric ion, with only a slight increase in the dissociation rate constant in the presence of Hg(II). Furthermore, the site of MerR binding to the DNA is in a novel position for a prokaryotic activator, directly between the two sets of recognition sequences for RNA polymerase. Analysis of the protein-DNA interactions and transcriptional activity has demonstrated that MerR forms a complex with RNA polymerase in the absence of Hg(II) that is unstable and transcriptionally repressed. When Hg(II) is present in greater than nanomolar concentrations, a highly active transcription complex is formed at PT and a distortion at the center of the palindromic MerR binding site is detectable. Kinetic analysis has determined that, although no change in the binding of RNAP to PT is apparent, the presence of Hg(II) stimulates the rate of isomerization from the closed to the open transcription complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metalloregulatory proteins and molecular mechanisms of heavy metal signal transduction. 220 24

Incubation of HeLa cells for 24 h with either hydroxyurea (HU), aphidicolin (APHI), thymidine (T) or butyrate (BU), substances used to inhibit replication and accumulate cells at the G1/S interphase, followed by the elimination of the inhibitor and the addition of iron to the growth medium, results in an immediate (HU, APHI, T) or slightly delayed (BU) increased accumulation (18-24-fold higher than the basal level) of ferritin. Under the same experimental circumstances, 5-azacytidine is without effect. As a result of the action of these inhibitors on the structure of DNA, it is proposed that ferritin genes remain accessible to RNA polymerase allowing the accumulation in the cytoplasm of mature ferritin mRNA ready to be mobilized by iron for the production of ferritin molecules.
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PMID:Stimulation of protein accumulation in HeLa cells by inhibitors of DNA replication. Ferritin. 241 93

The fhuB, fhuC and fhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm of Escherichia coli. The fhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence of fhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.
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PMID:Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane. 254 74


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