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)

Transcription of Adenovirus 2 DNA (Ad 2 DNA) by wheat germ RNA polymerase II in vitro satisfies criteria that have been used to establish that Escherichia coli or coliphage transcription in vitro is initiated at true promoters. (1) Wheat germ RNA polymerase forms highly stable complexes at specific sites on Ad 2 DNA, with a Kassoc of (4--5) X 10(10) M-1. (2) Electron microscopic visualization of enzyme bound to Ad 2 DNA reveals the location of eight strong binding sites, at least five of which appear to correspond to promoters that have been identified in studies of Ad 2 transcription in vivo [Evans, R. M., Fraser, N., Ziff, E., Weber, J., Wilson, M., & Darnell, J.E. (1977) Cell 12, 733--739; Berk, A.J., & Sharp, P.A. (1977) Cell 12, 45--55; Weinmann, R., & Aiello, L. O. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1662--1666]. (3) Transcription of Ad 2 DNA from preformed complexes with wheat germ polymerase is capable of escaping the action of rifamycin AF/013 and is relatively resistant to polyriboinosinic acid. In addition, our results are consistent with a two-state model for the interaction of wheat germ RNA polymerase with Ad 2 DNA, indicating that the mechansisms of transcription initiation and promoter-site selection in eucaryotes may be very similar to mechanisms elucidated in procaryotic systems.
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PMID:Interaction between wheat germ RNA polymerase II and adenovirus 2 DNA. Evidence for two types of stable binary complexes. 46 76

The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide-synthesizing enzymes were examined. Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases alpha, beta, gamma, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase. ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase alpha. Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template.primer for the same binding site of the enzyme. On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template.primers or nucleoside 5'-triphosphate substrates. The inhibition constants for ATA were, in general, smaller than those for Evans blue.
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PMID:Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid. 246 Mar 49

1. Labelled testosterone, injected directly into the ventral prostate of castrated rats became associated, in part, with a cytoplasmic high-molecular-weight fraction, fraction ;A'. 2. The label present in fraction ;A' was found to be mainly associated with dihydrotestosterone. 3. Unlike fraction ;A' from testosterone-pelleted castrated rats, fraction ;A' obtained from untreated castrated rats, 48h or more after castration, was strongly inhibitory towards Escherichia coli RNA polymerase in vitro. 4. The inhibition of RNA polymerase by fraction ;A' from castrated rats was not changed by the addition of testosterone or dihydrotestosterone in vitro, but pre-heating it to 80 degrees C resulted in a loss of its inhibitory capacity. 5. Fraction ;A' from castrated rats contained ribonuclease activity. The elution profile of ribonuclease activity from Sephadex columns indicated that this activity was responsible for the inhibitory effect on the RNA polymerase assays. 6. It is concluded that, unlike the inhibitor present in the uterus of ovariectomized rats (Talwar, Segal, Evans & Davidson, 1964), no direct connexion exists between the steroid-binding capacity of prostatic fraction ;A' and its effect on E. coli RNA polymerase activity in vitro.
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PMID:Some effects of testosterone on the rat ventral prostate. 424 60

Cutler, Richard G. (University of Houston, Houston, Tex.), and John E. Evans. Synchronization of bacteria by a stationary-phase method. J. Bacteriol. 91:469-476. 1966.-Cultures of Escherichia coli and Proteus vulgaris have been synchronized, with a high percentage phasing, in large volumes and at high cell densities by a method which takes advantage of a tendency of cells to synchronize themselves when entering the stationary phase of growth. The method consists of growing the bacteria to an early stationary phase, harvesting them quickly under minimal conditions of stress, and inoculating them into fresh medium at a dilution of about sevenfold. Cellular division is then partially synchronized. Four-generation cycles of a high percentage of phasing are obtained by repeating this procedure on the partially synchronized culture. Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein analyses were made throughout all phases of the growth curve. Advantage has been taken of this method of synchrony to isolate selected segments of the bacterial genome in significant amounts. A working hypothesis to explain the synchrony suggests that the unfavorable conditions of growth as the bacteria near the stationary phase are detected by a decrease in the amino acid pool size, and that this results in a gradual decrease of DNA transcription activity through the inhibition of RNA polymerase by transfer RNA. The synchronizing method may be unique in producing cultures that grow both in cellular division and in genomic synchrony.
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PMID:Synchronization of bacteria by a stationary-phase method. 532 75

DNA segments containing the major promoter at coordinate 16.5 for rightward transcription from human adenovirus serotypes 3 and 7 (Ad3 and Ad7), two closely related class B viruses, have been sequenced and found virtually identical. Furthermore, over 80% of the nucleotides of Ad3 and Ad7 in this entire region are homologous to their counterparts in the DNA of the more distantly related class C serotype Ad2. There are the same number of nucleotide pairs among these serotypes within the region compared. Most changes are transitions or transversions and the several single-base deletions are always compensated by nearby insertions. These few changes nonetheless result in 24 differences between Ad7 (or Ad3) and Ad2 in a total of 32 cleavage sites. The promoter for the rightward-transcribed RNAs and the first segment of the consanguinous tripartite leader found at the 5'-ends of all the later mRNAs derived from that promoter have been identified by analogy to the nucleotide sequences of Ad2. In particular, the "Hogness box" or RNA polymerase staging site for the major rightward transcription unit is completely homologous to that of Ad2. There are only six bp changes in the first late leader segment despite previous evidence suggesting that they might be quite heterologous. A prominent dyad axis of symmetry exists just upstream from the presumed 5'-end of the late RNA. However, unlike the stem-loop structure proposed for Ad2 by Ziff and Evans (1978), the base changes relative to Ad2 mandate a different potential stemloop structure in the single strand of Ad3 and Ad7 DNAs. This hairpin places the "Hogness box" immediately next to the 5'-end of the RNA at the base of stem. An analogous dyad axis of symmetry or stemloop structure can be found in a number of eukaryotic systems, including the major rightward transcription unit of Ad2. This feature may be of relevance to the positioning of RNA polymerase II on the DNA and to the promotion of transcription.
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PMID:Sequences of human adenovirus Ad3 and Ad7 DNAs encoding the promoter and first leader segment of late RNAs. 626 57

Cholecystokinin (CCK) has been suggested to modulate insulin output. We have shown that Otsuka Long-Evans Tokushima Fatty (OLETF) rats show little or no expression of the CCK-A receptor gene in the pancreas. We examined whether the CCK-A and CCK-B receptor genes are expressed in the islets and the role of CCK-A receptor in insulin secretion. Gene expressions of CCK receptors were determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization and Northern transfer analysis using LETO rats as controls. Pancreatic endocrine function was examined in perfusion (exogenous CCK stimulation) and meal ingestion (endogenous CCK stimulation) studies. CCK-A receptor mRNA was detected in the islets of LETO rats but not OLETF rats. Expression of the CCK-B receptor gene was detected in both strains by RT-PCR. Insulin secretion was impaired in OLETF rats, but the insulin contents of OLETF and LETO rats were not different. No abnormalities were detected histologically in either strain. These results suggest that the occurrence of pancreatic endocrine dysfunction in OLETF rats may be due to a defect in expression of the CCK-A receptor gene, not to insulin deficiency.
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PMID:Pancreatic endocrine dysfunction in rats not expressing the cholecystokinin-A receptor. 883 Mar 28

Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix associated with polymerase binding. The fluorescence of 2-aminopurine has been previously shown to depend on the environment of the base, with fluorescence increasing in the transition from a double-stranded to a single-stranded environment [Xu, D., Evans, K.O., & Nordlund, T. M.(1994) Biochemistry 33, 9592-9599]. Fluorescence changes associated with polymerase binding to promoters incorporating 2-aminopurine at positions -4 through -1 support a model which includes melting, in the statically bound complex, of the region of the promoter near the start site. Equilibrium titrations at 25 degrees C with label at position -2 provide a thermodynamic measure of the dissociation constant (Kd = 4.8 nM) for promoter binding, while stopped-flow kinetic assays measure the apparent association (k1 = 5.6 x 10(7) M-1 s-1) and dissociation (k-1 = 0.20 s-1) rate constants for simple promoter binding (the ratio k-1/k1 = 3.6 nM, in good agreement with the thermodynamic measurement of Kd). These results suggest that binding is close to the diffusion-controlled limit and helix melting is extremely rapid. In studies of structurally altered promoters, a base functional group substitution at position -10 is shown to significantly decrease k1, with little effect on k-1. In contrast, removal of the nontemplate strand from position +1 downstream results in a large decrease in k-1, with no significant effect on k1.
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PMID:Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase. 893 55

The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.
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PMID:Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay. 1075 4

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.
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PMID:Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. 1104 80

We have previously shown that the RNA polymerase 3D(pol) of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969-10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5' end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3D(pol) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359-10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2A(pro), serves as the primary template for HRV2 3D(pol) in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CD(pro). Our analyses suggest that HRV2 3D(pol) uses a "slide-back" mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2C(ATPase) coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A(1)A(2)A(3)CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.
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PMID:Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2A(pro). 1160 38


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