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 C-terminal domain (CTD) of RNA polymerase II (RNAP II) is essential for the assembly of RNAP II into preinitiation complexes on some promoters such as the dihydrofolate reductase (DHFR) promoter. In addition, during the transition from a preinitiation complex to a stable elongation complex, the CTD becomes heavily phosphorylated. In this report, interactions involving the CTD have been examined by protein-protein cross-linking. As a prelude to the study of CTD interactions, the effect of recombinant CTD on in vitro transcription was examined. The presence of recombinant CTD inhibits in vitro transcription from both the DHFR and adenovirus 2 major late promoters, suggesting that the CTD is involved in essential interactions with a general transcription factor(s). Factors in the transcription extract that interact with the CTD were identified by protein-protein cross-linking. Recombinant CTD was phosphorylated at its casein kinase II site, at the C terminus of the CTD, in the presence of [35S]adenosine 5'-O-(thiotriphosphate) and alkylated with azidophenacyl bromide. Incubation of azido-modified 35S-labeled CTD with a HeLa transcription extract followed by ultraviolet irradiation results in the covalent cross-linking of the CTD to proteins in contact with the CTD at the time of irradiation. Subsequent incubation with phenylmercuric acetate results in the transfer of 35S from the CTD to the protein to which it was cross-linked. The two major photolabeled bands have a M(r) of 34,000 and 74,000. The specificity of CTD interactions was demonstrated by a reduction in photolabeling in the presence of unmodified CTD or RNAP II containing an intact CTD (RNAP IIA) but not in the presence of a CTD-less RNAP II (RNAP IIB). The 35S-labeled 34- and 74-kDa proteins comigrate on SDS-polyacrylamide gel electrophoresis with the beta subunit of transcription factor IIE and the 74-kDa subunit of transcription factor IIF, respectively. Moreover, some of the minor 35S-labeled bands comigrate with other subunits of the general transcription factors.
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PMID:The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa cell transcription extract that comigrate with transcription factors IIE and IIF. 755 97

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.
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PMID:Identification of a poxvirus gene encoding a uracil DNA glycosylase. 838 53

Usefulness of MUC1 mRNA and keratin 19 mRNA as a target of reverse-transcriptase polymerase chain reaction (RT-PCR) was compared in the detection of breast cancer micrometastases in axillary lymph nodes. RT-PCR amplification of MUC1 mRNA and keratin 19 mRNA was conducted using total RNA samples. RT-PCR products were stained with ethidium bromide and analyzed by agarose gel electrophoresis. Expression of both MUC1 mRNA and keratin 19 mRNA was detected by RT-PCR in a breast cancer cell line (MRK) and in all the 23 primary breast cancers but not in the control lymph nodes obtained from patients with benign diseases. A serial dilution study of MRK cells against normal lymph node cells has shown that detection sensitivity of MUC1 RT-PCR and keratin 19 RT-PCR were 1/10(5) and 1/10(6) (cancer/lymph node cells), respectively. Sixty-three axillary lymph nodes were obtained from 23 patients with primary breast cancer, and metastases in each lymph node were investigated by histological examination (hematoxylin and eosin sections) and RT-PCR method. In all 10 lymph nodes, which were histologically metastasis-positive, both MUC1 mRNA and keratin mRNA were detected by RT-PCR. Of the 53 histologically negative lymph nodes, 3 (6%) and 5 (9%) lymph nodes were found to express MUC1 mRNA and keratin 19 mRNA, respectively, indicating the presence of micrometastases which could be detected by RT-PCR but not by histological examination. These results demonstrate the usefulness of both MUC1 RT-PCR and keratin 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes, and also indicate the superiority of keratin 19 RT-PCR over MUC1 RT-PCR because of its higher detection sensitivity.
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PMID:Detection of breast cancer micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Comparison between MUC1 mRNA and keratin 19 mRNA amplification. 857 27

A reverse transcriptase activity has been detected in potato mitochondria using special RNAs as templates: a bacterial RNA coding for neomycin phosphotransferase (neo pa RNA) and a Neurospora crassa mitochondrial RNA (184 nt RNA). Surprisingly, no exogenous primer addition was required. These RNA templates share a primary and secondary structure similar to the T psi CG loop of tRNAs that could constitute the recognition site for the enzyme. Reverse transcriptase activity was inhibited by ddTTP, ethidium bromide and aphidicolin, while potato mitochondrial DNA polymerase was not inhibited by aphidicolin indicating that these activities correspond to distinct enzymes. A conserved sequence of reverse transcriptases was detected in potato mitochondrial DNA suggesting that this enzyme could be mitochondrially encoded.
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PMID:A reverse transcriptase activity in potato mitochondria. 875 99

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.
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PMID:A NusG-like protein from Thermotoga maritima binds to DNA and RNA. 876 36

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.
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PMID:Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm. 891 43

Brome mosaic bromovirus (BMV), a positive-stranded RNA virus, supports both homologous and nonhomologous RNA recombinations. Two BMV (temperature-sensitive) mutants with alterations in the 2a protein, the putative RNA polymerase component of the viral replicase, were tested for their ability to support both types of recombination. Here we report that one of these mutants with the Leu-486 substituted by Phe did not support nonhomologous recombination. Effect on homologous recombination was mainly on the location and precision of crossover events. The other 2a mutant with Asn-458 substituted by Asp did not negatively affect either type of recombination. Apparently, BMV RNA polymerase participates differently in the two types of recombination events.
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PMID:A mutation in the putative RNA polymerase gene inhibits nonhomologous, but not homologous, genetic recombination in an RNA virus. 905 Sep 7

We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.
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PMID:Effects of neutral salts on RNA chain elongation and pausing by Escherichia coli RNA polymerase. 914 40

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


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