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)

Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.
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PMID:A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF. 1150 14

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.
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PMID:The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. 1191 76

We report the complete sequence of a large rod-shaped DNA virus, called the Hz-1 virus. This virus persistently infects the Heliothis zea cell lines. The Hz-1 virus has a double-stranded circular DNA genome of 228,089 bp encoding 154 open reading frames (ORFs) and also expresses a persistence-associated transcript 1, PAT1. The G+C content of the Hz-1 virus genome is 41.8%, with a gene density of one gene per 1.47 kb. Sequence analysis revealed that a 9.6-kb region at 43.6 to 47.8 map units harbors five cellular genes encoding proteins with homology to dUTP pyrophosphatase, matrix metalloproteinase, deoxynucleoside kinase, glycine hydroxymethyltransferase, and ribonucleotide reductase large subunit. Other cellular homologs were also detected dispersed in the viral genome. Several baculovirus homologs were detected in the Hz-1 virus genome. These include PxOrf-70, PxOrf-29, AcOrf-81, AcOrf-96, AcOrf-22, VLF-1, RNA polymerase LEF-8 (orf50), and two structural proteins, p74 and p91. The Hz-1 virus p74 homolog shows high structural conservation with a double transmembrane domain at its C terminus. Phylogenetic analysis of the p74 revealed that the Hz-1 virus is evolutionarily distant from the baculoviruses. Another distinctive feature of the Hz-1 virus genome is a gene that is involved in insect development. However, the remainder of the ORFs (81%) encoded proteins that bear no homology to any known proteins. In conclusion, the sequence differences between the Hz-1 virus and the baculoviruses outnumber the similarities and suggest that the Hz-1 virus may form a new family of viruses distantly related to the Baculoviridae:
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PMID:Analysis of the complete genome sequence of the Hz-1 virus suggests that it is related to members of the Baculoviridae. 1218 86

Amino acids at conserved sites in the residue sequence of 10 ancient proteins, from 844 phylogenetically diverse sources, were used to specify their time of origin in the interval before species divergence from the last common ancestor (LCA). The order of amino acid addition to the genetic code, based on biosynthesis path length and other molecular evidence, provided a reference for evaluating the 'code age' of each residue profile examined. Significantly earlier estimates were obtained for conserved amino acid residues in these proteins than non-conserved residues. Evidence from the primary structure of 'fossil' proteins thus corroborated the biosynthetic order of amino acid addition to the code.Low potential ferredoxin (Fdxn) had the earliest residue profile among the proteins in this study. A phylogenetic tree for 82 prokaryote Fdxn sequences was rooted midway between bacteria and archaea branches. LCA Fdxn had a 23-residue antecedent whose residue profile matched mid-expansion phase codon assignments and included an amide residue. It contained a highly acidic N-terminal region and a non-charged C-terminal region, with all four cysteine residues. This small protein apparently anchored a [4Fe-4S] cluster, ligated by C-terminal cysteines, to a positively charged mineral surface, consistent with mediating e(-) transfer in a primordial surface system before cells appeared. Its negatively charged N-terminal 'attachment site' was highly mutable during evolution of ancestral Fdxn for Bacteria and Archaea, consistent with a loss of function after cell formation. An initial glutamate to lysine substitution may link 'attachment site' removal to early post-expansion phase entry of basic amino acids to the code. As proteins evidently anchored non-charged amide residues initially, surface attachment of cofactors and other functional groups emerges as a general function of pre-cell proteins.A phylogenetic tree of 107 proteolipid (PL) helix-1 sequences from H(+)-ATPase of bacteria, archaea and eukaryotes had its root between prokaryote branches. LCA PL h1 residue profile optimally fit a late expansion phase codon array. Sequence repeats in transmembrane PL helices h1 and h2 indicated formation of the archetypal PL hairpin structure involved successive tandem duplications, initiated within the gene for an 11-residue (or 4-residue) hydrophobic peptide. Ancestral PL h1 lacked acidic residues, in a fundamental departure from the prototype pre-cell protein. By this stage, proteins with a hydrophobic domain had evolved. Its non-polar, late expansion phase residue profile point to ancestral PL being a component of an early permeable cell membrane. Other indicators of cell formation about this stage of code evolution include phospholipid biosynthesis path length, FtsZ residue profile, and late entry of basic amino acids into the genetic code. Estimates based on conserved residues in prokaryote cell septation protein, FtsZ, and proteins involved with synthesis, transcription and replication of DNA revealed FtsZ, ribonucleotide reductase, RNA polymerase core subunits and 5'-->3' flap exonuclease, FEN-1, originated soon after cells putatively evolved. While reverse transcriptase and topoisomerase I, Topo I, appeared late in the pre-divergence era, when the genetic code was essentially complete. The transition from RNA genes to a DNA genome seemingly proceeded via formation of a DNA-RNA heteroduplex. These results suggest formation of DNA awaited evolution of a catalyst with a hydrophobic domain, capable of sequestering radical bearing intermediates in its synthesis from ribonucleotide precursors. Late formation of topology altering protein, Topo I, further suggests consolidation of genes into chromosomes followed synthesis of comparatively thermostable DNA strands.
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PMID:Molecular evolution before the origin of species. 1222 77

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe, encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed.
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PMID:MCB-mediated regulation of cell cycle-specific cdc22+ transcription in fission yeast. 1289 17

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.
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PMID:Possible antitumor activity of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) against an established gemcitabine (dFdCyd)-resistant human pancreatic cancer cell line. 1590 71

Novel N-1-sulfonylpyrimidine derivatives have a strong antiproliferative activity and an ability to induce apoptosis in treated tumor cells. The purpose of this study was to elucidate the effects of two N-1-sulfonylpyrimidine nucleobases on catalytic activity of tumor cells' enzymes involved in DNA and RNA synthesis, and in de novo and salvage pyrimidine and purine syntheses. Investigations were performed in vitro on colon carcinoma cells (Caco2). The biosynthetic activity of the tumor cells' enzymes was determined using sensitive radio-assays. Enzyme activity in treated cells was calculated relative to untreated control cells. Both of the investigated compounds, 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) inhibited activities of specific enzymes involved in nucleic acid synthesis. BMsU strongly inhibited activities of DNA polymerase alpha (53%), thymidine kinase (68%), thymidilate synthase (43%), and ribonucleotide reductase (46%). De novo biosynthesis of pyrimidine and purine was reduced by 20%. TsC was able to inhibit RNA polymerase (37%), orotate phosphoribosyltransferase (39%), uridine kinase (44%), ribonucleotid reductase (47%), and de novo purine synthesis (61%). Antitumor activity of 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) is closely associated with their inhibitory activity on enzymes that play an important role in the metabolism of tumor cells.
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PMID:Metabolic effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma cells. 1591 14

Genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU) induced replication stress and ionizing radiation in yeast. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4 and degradation of its inhibitor, Sml1p. The Ccr4-Not complex has originally been described as a negative regulator of RNA polymerase II (pol II) transcription, but it has also been implicated in mRNA turnover and protein ubiquitination. We investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU induced Sml1p degradation. We show, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or methyl-methane sulfonate (MMS) treatment. Chromatin immunoprecipitation (ChIP) experiments show that TBP, pol II and Set1p recruitment to the activated RNR3 locus is defective in cells lacking NOT4. Moreover, RNR3-promoter activity is not induced by HU in these cells. Our experiments show that induction of RNR gene transcription is defective in ccr4-not mutant strains, providing an explanation for their sensitivity to HU.
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PMID:DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4-Not complex. 1627 85

Two bacteriophages, DSS3Phi2 and EE36Phi1, which infect marine roseobacters Silicibacter pomeroyi DSS-3 and Sulfitobacter sp. EE-36, respectively, were isolated from Baltimore Inner Harbor water. These two roseophages resemble bacteriophage N4, a large, short-tailed phage infecting Escherichia coli K12, in terms of their morphology and genomic structure. The full genome sequences of DSS3Phi2 and EE36Phi1 reveal that their genome sizes are 74.6 and 73.3 kb, respectively, and they both contain a highly conserved N4-like DNA replication and transcription system. Both roseophages contain a large virion-encapsidated RNA polymerase gene (> 10 kb), which was first discovered in N4. DSS3Phi2 and EE36Phi1 also possess several genes (i.e. ribonucleotide reductase and thioredoxin) that are most similar to the genes in roseobacters. Overall, the two roseophages are highly closely related, and share 80-94% nucleotide sequence identity over 85% of their ORFs. This is the first report of N4-like phages infecting marine bacteria and the second report of N4-like phage since the discovery of phage N4 40 years ago. The finding of these two N4-like roseophages will allow us to further explore the specific phage-host interaction and evolution for this unique group of bacteriophages.
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PMID:Genome sequences of two novel phages infecting marine roseobacters. 1968 6

Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, prevents cells from progressing through S phase by depletion of deoxyribonucleoside triphosphates. Concurrently, disruption of DNA replication leads to double-strand DNA breaks. In root meristems of Vicia faba, HU triggers cell cycle arrest (preferentially in G1/S phase) and changes an overall metabolism by global activation of transcription both in the nucleoplasmic and nucleolar regions. High level of transcription is accompanied by an increase in the content of RNA polymerase II large subunit (POLR2A). Changes in transcription activation and POLR2A content correlate with posttranslational modifications of histones that play a role in opening up chromatin for transcription. Increase in the level of H4 Lys5 acetylation indicates that global activation of transcription following HU treatment depends on histone modifications.
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PMID:Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba. 2252 1


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