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)

Sp1 is a sequence-specific DNA binding protein that activates RNA polymerase II transcription from promoters that contain properly positioned GC boxes. A series of deletion mutants of Sp1 were expressed in Escherichia coli and used to identify separate regions of the protein that are important for three different biochemical activities. The sequence-specificity of DNA binding was conferred by Zn(II) fingers, whereas a different region of Sp1 appeared to regulate the affinity of DNA binding. The E. coli-synthesized Sp1 was able to stimulate initiation of RNA synthesis in vitro, and at least two distinct segments of the protein contributed to its transcriptional activity.
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PMID:Distinct regions of Sp1 modulate DNA binding and transcriptional activation. 305 95

T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.
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PMID:Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347. 308 55

DNA-dependent RNA polymerase (RPase) from Escherichia coli contains 2 mol of intrinsic Zn(II)/mol of core enzyme (alpha 2 beta beta'). In techniques analogous to those employed with the Zn(II) metalloenzyme aspartate transcarbamoylase [Hunt, J. B., Neece, S. H., Schachman, H. K., & Ginsberg, A. (1984) J. Biol. Chem. 259, 14793-14803], we show that titration of core or holoRPase with 10 or 16 equiv, respectively, of the sulfhydryl reagent p-(hydroxymercuri)benzenesulfonate (PMPS) results in the facile release of 1 mol of Zn(II) [B-site Zn(II)] in a reaction totally reversible with the addition of excess thiol provided no metal chelator is present. If ethylenediaminetetraacetic acid (EDTA) is present, reversal of the PMPS-enzyme complex results in formation of a Zn1 RPase [A-site Zn(II)]. This enzyme retains full transcriptional activity relative to Zn2 RPase on both calf thymus (nonspecific) and T7 (sigma-dependent, specific) DNA templates. If the core enzyme-PMPS complex is incubated with a large excess of another metal such as Cd(II) followed by thiol treatment, a hybrid ZnACdB RPase is formed. Direct treatment of the enzyme with excess Cd(II) also gives rise to a hybrid ZnACdB RPase. Transcription by these enzymes is also comparable to that of the starting Zn2 enzyme. Isolation of in vivo synthesized Co2 RPase and Cd2 RPase and treatment of either enzyme with PMPS/EDTA results in formation of a CoA and CdA enzyme, respectively. Co(II)A and Cd(II)A enzymes show 123 and 76%, respectively, of the elongation rates on T7 DNA observed for the Zn(II) enzyme. Visible absorption spectroscopy of the Co2 enzyme exhibits four d-d transition bands positioned at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. In addition, two charge-transfer bands are found at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. Only the Co(II) ion bound at site A is associated with this unique set of intense d-d transitions. The positions and intensities of both the visible and charge-transfer bands of Co(II)A RPase approximate those shown by Co(II)-substituted metalloenzyme sites where the ligands are four S rather than mixed S,N or S,O sites.
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PMID:Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase. 309 79

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.
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PMID:DNA-dependent RNA polymerase from Pseudomonas aeruginosa. 312 44

Metallothioneins that bind copper and zinc have an Mr of 6500 daltons, consist of a single polypeptide chain of 61 amino acids, 25-30 percent of whose residues are cysteine, have a metal-binding capacity of between 5 and 7 g atoms/mol, and contain no disulfide bonds or aromatic amino acids. Zincthionein has been postulated to participate in the transport and storage of zinc, which is involved in more than 235 metalloenzymes, including thymidine kinase, RNA polymerase, and ribonuclease, which in turn play crucial roles in the replication and transcription of DNA during cell division. In addition, trace elements including zinc modulate immune response and function. Conversely, zinc deficiency state causes, for example, thymic atrophy and lymphopenia and modifies antibody-mediated responses to both T-cell-dependent and T-cell-independent antigens. The concentrations of copper, zinc, and metallothionein and the copper/zinc ratio are modified in a number of malignancies. For example, the levels of metallothionein in normal and in malignant human livers are 471 and 75 micrograms/g, respectively. In addition, the copper/zinc ratio is significantly increased in human pancreatic cancer from 1.40 to 2.70. Furthermore, studies involving 64Cu in tumor-bearing mice showed that the distribution of 64Cu was altered and that all tumors contained a relatively high level of 64Cu. Moreover, the activity of superoxide dismutase to remove free oxygen radicals is lower in malignant tissues. Finally, the results of clinical studies suggest that the monitoring of the serum copper/zinc ratio may be a valuable tool, not only in determining the extent of malignancies, but also in predicting the efficacy of treatments.
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PMID:The status of zinc, copper, and metallothionein in cancer patients. 328 43

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.
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PMID:Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae. 329 61

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.
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PMID:Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. 330 70

RNA polymerase (RPase) from E. coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay. One of the two Zn(II) ions can be differentially removed from E. coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol. The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters. Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. The absorption characteristics strongly suggest that the A site is a tetrathiolate site. While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II). PMPS displaces the zinc by reacting with three SH groups. Apo-g32P shows markedly altered DNA binding properties. Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm. The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P. Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis.
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PMID:Zinc metalloproteins involved in replication and transcription. 354 19

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.
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PMID:Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. 354 6

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. 354 18


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