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)

The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - sigma H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53 degrees C.
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PMID:Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis. 174 26

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.
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PMID:A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258. 193 59

The effects of 14 metal ions (chlorides) on the transcription of calf thymus DNA and phage T4 DNA with Escherichia coli RNA polymerase were tested. These assays were conducted under improved conditions of lower pH and in the absence of 2-mercaptoethanol to permit greater stability of the metal ions in solution. Among the divalent metal ions tested, the concentration-dependent order of inhibition of overall transcription is Pb2+ greater than Zn2+ greater than Cu2+ greater than Be2+ greater than Cd2+ greater than Ni2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Sr2+ and is the same with either template. At pH 7.4 and in the absence of 2-mercaptoethanol, considerably lower concentrations of several of the divalent metal ions are needed for inhibition of overall transcription than at pH 8.1 and in the presence of 2-mercaptoethanol. Ca2+, Mg2+, Sr2+, Zn2+, Li+, Na+, and K+--considered to be non-mutagenic and non-carcinogenic--decrease chain initiation (measured with T4 DNA) at concentrations that inhibit overall transcription. Pb2+, Cd2+, Co2+, Be2+, and Mn2+--all mutagenic or carcinogenic--stimulate chain initiation (although at widely different rates) at concentrations that inhibit overall transcription. Cu2+ and Ni2+--both carcinogenic--stimulate initiation only at very low concentrations, followed by a progressive decrease in initiation at concentrations that inhibit overall transcription.
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PMID:Selective effects of metal ions on RNA synthesis rates. 617 51

By use of poly(dA-dT) as template and Escherichia coli RNA polymerase, several metal ions were tested for their effect on the efficiency of transcription and on the misincorporation of CMP into the poly(rA-rU) product. In the presence of 10 mM MgCl2, Mn2+ has a stimulatory effect on the transcription, Co2+ has very little effect on the reaction, Cu2+ and Zn2+ are strongly inhibitory, and Cd2+ and Ni2+ are less inhibitory. The background misincorporation of CMP in the presence of MgCl2 is about 1 nucleotide per 2000 correct nucleotides incorporated and is independent of Mg2+ concentration. Zn2+, Ca2+, Sr2+, Li+, Na+, and K+--all nonmutagenic and noncarcinogenic--do not increase misincorporation. Mn2+ causes a concentration-dependent threefold increase in the misincorporation that can be slightly reversed at higher MgCl2 concentrations. Cd2+ causes a dramatic increase in the misincorporation with increasing CdCl2 concentration that can be substantially overcome by higher concentrations of Mg2+. Cu2+ also increases the misincorporation, Ni2+ slightly increases it, and Co2+ does not increase it at all. Several control experiments indicate that the misincorporation of CMP is dependent on the template-directed synthesis of poly(rA-rU). Nearest-neighbor analysis indicates that CMP is incorporated in place of UMP into the poly(rA-rU) product. The increase in misincorporation appears to be related both to the "hard-soft" character of the metal ions and to their carcinogenic potential.
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PMID:Effect of several metal ions on misincorporation during transcription. 702 4

The CadC protein from the cadA cadmium resistance operon of Staphylococcus aureus plasmid pI258 regulates transcription of this system in vitro. The CadC protein was overproduced in Escherichia coli cells and partially purified. Gel shift assays of the proposed cadA operator/promoter region DNA showed specific association with the CadC protein. Control arsenic resistance operator/promoter DNA from the same plasmid was not shifted by the CadC protein. Cd2+, Bi3+, and Pb2+ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions -7 to +14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.
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PMID:CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258. 754 76

Introduction of the cadmium chloride water solution to experimental animals induces changes in biochemical parameters which characterize structural and functional activity of transcriptionally active and repressed chromatin fractions. In the intoxicated chromatin-active fraction the DNA/protein ratio increases and DNA-polymerase alpha-activity decreases while in repressed chromatin activity of RNA polymerase I decreases as compared with controls. Change in intensity of lipoperoxidation reactions may underlie the cadmium chloride genotoxicity. This thesis is proved by an augmented level of NADPH-induced lipoperoxidation in active chromatin fraction.
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PMID:[Effect of cadmium chloride on DNA-, RNA-polymerase activity and lipid peroxidation of chromatin fraction in rat liver]. 816 Feb 91

Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif. The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (delta epsilon 248, 24 mM-1 cm-1) indicated the presence of a Cd(Cys-S)4 center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD, app 3-4 microM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between-70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase alpha-subunit. A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.
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PMID:Metal- and DNA-binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: a prokaryotic C4 zinc-finger protein. 909 99

1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.
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PMID:Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. 950 29

Xenopus upstream binding factor (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. Among these DNA-binding motifs, HMG box I is essential for promoting RNA polymerase I-dependent rRNA gene transcription. Gel shift assay indicated that the binding of recombinant HMG box I to a 136-bp linear DNA probe was significantly inhibited by Cd2+ at 1 microM. The formation of larger protein-DNA complexes was particularly sensitive to Cd2+. The interaction between HMG box I and DNA was completely inhibited by 10 microM of Cd2+, yet this interaction was not inhibited by the same concentration of Ca2+. Hg2+ at 0.1 microM began to cause abnormal band shifting, and protein-DNA bands disappeared to the wells of a polyacrylamide gel in the presence of 10 microM of Hg2+, reflecting that a drastic change in the conformation of HMG box I-DNA had occurred. The binding of HMG box I to DNA was slightly disturbed by As3+ at 1 microM and was significantly affected at 10 microM. Our results suggest that inhibition of the normal binding of UBF to its target DNA may be one of the mechanisms of heavy metal-induced inhibition of RNA synthesis.
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PMID:Differential effects of heavy metals on the binding of Xenopus upstream binding factor(xUBF) to DNA. 956 4

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.
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PMID:Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels. 960 Oct 77


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