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)

Promoter escape can be rate-limiting for transcription by bacterial RNA polymerases and RNA polymerase II of higher eukaryotes. Formation of a productive elongation complex requires disengagement of RNA polymerase from promoter-bound eukaryotic transcription factors or bacterial sigma factors. RNA polymerase III (pol III) stably associates with the TFIIIB-DNA complex even in the absence of localized DNA unwinding associated with the open promoter complex. To explore the role that release of pol III from the TFIIIB-DNA complex plays in limiting the overall rate of transcription, we have examined the early steps of RNA synthesis. We find that, on average, only three rounds of abortive initiation precede the formation of each elongation complex and that nearly all pol III molecules escape the abortive initiation phase of transcription without significant pausing or arrest. However, when elongation is limited to 5 nucleotides, the intrinsic exoribonuclease activity of pol III cleaves 5-mer RNA at a rate considerably faster than product release or reinitiation. This cleavage also occurs in the normal process of forming a productive elongation complex. The possible role of nucleolytic retraction in disengaging pol III from TFIIIB is discussed.
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PMID:Abortive initiation by Saccharomyces cerevisiae RNA polymerase III. 1047 18

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K(D)) for binding of MerR were: binding site I, 8.5 x 10(-9) M; binding site II, 1.2 x 10(-8) M; and for the complete promoter/operator region 1 x 10(-8) M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K(D) value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 x 10(-7) M.
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PMID:Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326. 1050 47

We report the discovery and characterization of the mercury resistance transposon, Tn5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn5044 is expressed at low (30 degrees C) but not at elevated temperatures (37 degrees C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn5044 is closely related to those of Tn1412 isolated from medical sources and to Tn5563 and ISXc5 from environmental sources. However, Tn5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase (tnpA) genes places Tn5044 and its close relatives into the Tn3 subgroup of the Tn3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn5044 resolvase can resolve cointegrates formed during Tn5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.
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PMID:Tn5044, a novel Tn3 family transposon coding for temperature-sensitive mercury resistance. 1087 86

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11-amino acid residue peptide from the C-terminus of adenovirus precursor protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.
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PMID:Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA. 1168 32

Puumala (PUU) virus causes a form of hemorrhagic fever with renal syndrome (HFRS), called nephropathia epidemica (NE), in Europe. HFRS is characterized by an increased capillary permeability, which we hypothesize is caused by hyperactivation of the host immune system, especially cellular immune responses. To identify cytotoxic T lymphocytes (CTLs) specific for the PUU virus from NE patients, we have made recombinant vaccinia viruses expressing PUU virus proteins, the nucleocapsid (N) and two surface glycoproteins, G1 and G2. Recombinant vaccinia viruses carrying the N or the first half of the G2 cDNA under the control of a strong synthetic promoter were made. To express G1 and the second half of the G2 proteins, however, we needed to use a T7 expression system, where the T7 RNA polymerase is produced from another recombinant vaccinia virus co-infecting the same cells. These recombinant vaccinia viruses were used to detect and clone PUU virus-specific CTLs from the peripheral blood mononuclear cells of NE patients. An HLA-A24-restricted CTL line recognizing the G2 protein was isolated and its 9-mer epitope was determined.
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PMID:Generation of recombinant vaccinia viruses expressing Puumala virus proteins and use in isolating cytotoxic T cells specific for Puumala virus. 1190 Aug 40

The aim of this study was to identify the B cell epitopes of hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase (RdRp). The truncated HCV NS5B protein NS5B-dc21 was expressed in Escherichia coli and its antigenicity was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) using 130 HCV-positive human sera and 15 negative sera. Antibodies specific to NS5B-dc21 protein were purified by affinity chromatography using sepharose-4B coupled with the recombinant protein. A 12-mer phage displayed random peptide library was screened four rounds with the purified antibodies. Three epitopes were identified from the phage library, which correspond to amino acids 2444-2452, 2521-2528, and 2915-2925 of HCV RdRp. These epitopes were then expressed in E. coli as fusion proteins with phage M13 pIII protein. ELISA demonstrated that two of these epitopes (P4 and P34, corresponding to amino acids 2443-2452 and amino acids 2512-2528, respectively) have good reactivity and sensitivity. Mutagenesis study of P4 peptide showed that this epitope, which is derived from a phage displayed library, exhibited higher affinity with HCV serum than the corresponding original HCV sequences.
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PMID:Identification of B cell epitopes of hepatitis C virus RNA dependent RNA polymerase. 1202 Jul 87

Recent genetic data suggest that proteins homologous to a plant RNA-dependent RNA polymerase (RdRP) play a central role in posttranscriptional gene silencing (PTGS) in many organisms. We show here that purified recombinant protein QDE-1, a genetic component of PTGS ("quelling") in the fungus Neurospora crassa, possesses RNA polymerase activity in vitro. The full-length enzyme and its enzymatically active C-terminal fragment perform two different reactions on single-stranded RNA templates, synthesizing either extensive RNA chains that form template-length duplexes or approximately 9-21-mer complementary RNA oligonucleotides scattered along the entire template. QDE-1 supports both de novo and primer-dependent initiation mechanisms. These results suggest that several distinct activities of cell-encoded RdRPs can be employed for efficient PTGS in vivo.
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PMID:Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. 1250 16

Vaccinia virus early gene transcription termination requires the vaccinia termination factor (VTF), NPH I, a single stranded DNA-dependent ATPase, the virion form of RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU, which resides in the nascent mRNA, located 30 to 50 bases upstream from the poly(A) addition site. Evidence indicates that a required termination factor acts through binding to the UUUUUNU signal. To further investigate the function of UUUUUNU, the ability of UUUUUNU containing oligonucleotides to inhibit transcription termination was tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature transcription termination and transcript release, in trans. Activation of premature termination required VTF, NPH I, Rap 94, and ATP, demonstrating that the normal termination machinery was employed. Premature termination was not stimulated by RNA harboring a mutant UUUUUNU, demonstrating specificity. These data are consistent with a model in which a required termination factor is converted from an inactive to an active form by binding to a UUUUUNU containing oligonucleotide. The active termination factor then interacts with the ternary complex stimulating transcription termination through the normal mechanism, independent of the nascent mRNA sequence.
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PMID:UUUUUNU oligonucleotide stimulation of vaccinia virus early gene transcription termination, in trans. 1255 20

A non-natural beta-C-nucleoside bearing a 3,4-dibenzyloxyphenyl group as a nucleobase (X) was synthesized and incorporated into a 34-mer oligomer with the sequence 5'-dTTTTTAAAAAAXATATAGCAGCGACATGTCACCG-3'. This synthetic oligonucleotide was examined for template activity in the enzymatic syntheses of DNA by the Klenow fragments of Escherichia coli DNA polymerase I and the recombinant DNA polymerase I, and in the synthesis of RNA by the E. coli RNA polymerase core enzyme. As a result, the template-directed polymerization of both DNA and RNA was precisely terminated at the position of X. The X-containing oligonucleotide was also tested for digestion by an exonuclease, Exo III nuclease (Exo III), and an endonuclease, Mung Bean nuclease (MB). The results indicate that the artificial nucleobase X acts as a terminator for digestion by Exo III, whereas the site X becomes susceptible to digestion by MB. These findings provide a useful tool for the size control of products in the synthesis and degradation of nucleic acids.
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PMID:Role of a non-natural beta-C-nucleotide unit in DNA as a template for DNA and RNA syntheses and as a substrate for nucleolytic digestion. 1367 92

The major challenges for anti-hepatitis B Virus (HBV) therapy are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. A drug with new target sites or independent metabolic pathways may overcome these shortcomings. Small interfering RNA (siRNA) offers the possibility of developing a new anti-HBV therapy. Here we describe the almost complete inhibition of HBV replication by stably expressed 21-mer short hairpin RNAs (shRNA). Besides the conventional targets on HBV reverse-transcriptase, we also systemically targeted other sites of pregenomic RNA, including direct repeat (DR) elements, S, core, and X gene. Our results indicated that shRNAs can serve as efficient alternative anti-HBV agents. They can also be used in combination with chemotherapy, because they showed better effects on the inhibition of HBV replication due to different mechanisms of drug actions.
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PMID:Inhibition of hepatitis B virus replication by stably expressed shRNA. 1459 28


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