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 sugar boronated thymidine nucleoside, 5' -0-[(triphenylphosphine-boryl) carbonyl]-3'-0-acetyl thymidine 1, and the boron-modified nucleoside phosphotriester, 5'-(diethylphosphite- cyanoborane)-3'-acetylthymidine 2, were successfully synthesized. Both compounds demonstrated differential activity when tested against eight cell lines, with significant cytotoxic activity against the growth of human Tmolt3 leukemia, colon adenocarcinoma, HeLa S3 uterine carcinoma, and osteosarcoma cells. In in vivo studies these agents were found to be active against the growth of Ehrlich ascites carcinoma at 8 mg/kg/day I.P. and to be marginally active against the growth of L1210 and Lewis lung cancers in mice. The mode of action of these thymidine derivatives in Tmolt3 cells was the inhibition of DNA and protein synthesis. Compound 2 was highly effective in inhibiting DNA polymerase alpha and m-RNA, r-RNA and t-RNA polymerase activities. Both compounds inhibited ribonucleoside reductase activity. The de novo purine pathway appeared to be the major site of inhibition of the agents, with IMP dehydrogenase, PRPP amido transferase, and dihydrofolate reductase activities being significantly inhibited. In the pyrimidine pathway, carbamyl phosphate synthetase and aspartate transcarbamylase activities were inhibited by 1. As expected, d[NTP] levels were significantly reduced by treatment with the agents. DNA strand scission was evident after incubating Tmolt3 cells for 24 hr with the agents.
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PMID:Antineoplastic activity of boron-containing thymidine nucleosides in Tmolt3 leukemic cells. 150 1

The transcriptional commitment of mitochondrial RNA (mtRNA) polymerase and the conditions required for the formation of a stable ternary complex have been determined by in vitro transcription study. Four different transcription complexes were made in vitro by incubating purified mtRNA polymerase, cloned synthetic mitochondrial promoters and selective ribonucleotides. The responses of these complexes to heparin, an inhibitor of unbound mtRNA polymerase, have been examined to determine their involvement in transcription. This study leads to the following observations. (1) Under normal reaction conditions, 40 nM-heparin completely inhibited mitochondrial transcription. (2) A preinitiation mitochondrial DNA-RNA polymerase complex (complex 0) showed partial resistance to heparin (approximately 25% resistant to 40 nm-heparin) when heparin and ribonucleoside triphosphates (rNTPs) were added together to the preformed complex. This complex was rapidly inactivated when preincubated with heparin before the addition of rNTPs. (3) The early initiation (complexes 2 and 4) containing DNA template, RNA polymerase and a short RNA product showed more resistance (approx. 40 to 50%) to 40 nM-heparin but destabilized upon further incubation with heparin before addition of the rest of the rNTPs. (4) After generation of ten or more phosphodiester bonds (complex 11), the early transcription complex is converted into a stable initiation complex, leading to the polymerase consignment to elongation. On the basis of stability and heparin sensitivity, three initial steps of mitochondrial transcription have been defined: polymerase-promoter interaction, initiation, and the transition from initiation to elongation. The formation of preinitiation complex is the rate-limiting step t 1/2 approx. 50 s), whereas the initiation and elongation reactions are very fast processes (t 1/2 greater than 5 s) in mitochondrial transcription.
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PMID:Transcriptional commitment of mitochondrial RNA polymerase from Saccharomyces cerevisiae. 164 Apr 54

Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain. Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end. The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA. The photo-cross-linking nucleotide analog used for these studies was 5-[4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe. With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted. When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted. No labeling of the sigma or alpha subunits was observed with any RNA length. In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts. Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream. These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter. This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP. By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.
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PMID:Active site labeling of Escherichia coli transcription elongation complexes with 5-[4-azidophenacyl)thio)uridine 5'-triphosphate. 169 25

An in vitro system allowing human cell extracts to carry out excision repair in ultraviolet light-irradiated, closed circular DNA was used to study the influence of transcription on repair synthesis. RNA synthesis from plasmid DNA containing bacterial transcription units was obtained by addition of Escherichia coli RNA polymerase and ribonucleoside triphosphates to the repair-incubation mixture. No increase in UV-stimulated repair replication was observed under transcriptional conditions; in fact, an inhibition of repair replication occurred, possibly due to impairment of DNA polymerization. This is unlike the in vivo situation where preferential DNA repair of transcribed genes has been found after UV-irradiation. Possible factors important for preferential repair of expressed genes in mammalian cells are discussed.
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PMID:Influence of RNA synthesis on DNA-repair replication in human cell extracts. 169 81

We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [alpha-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.
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PMID:RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage. 170 18

RNA-based amplification systems that have been recently described are dependent upon the presence of more than one enzyme. In an attempt to minimize the number of polymerases required for efficient amplification, we have studied the template specificity of bacteriophage T3 RNA polymerase. A synthetic bacteriophage T3 promoter was covalently attached to an RNA template. The T3 promoter-RNA complex was found to be selective for its native polymerase, and dependent upon the presence of all four ribonucleoside precursors. The product of the RNA-directed transcription is complementary to the initial template.
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PMID:DNA-dependent RNA polymerase from bacteriophage T3 transcribes and amplifies an RNA template in vitro. 171 21

Phosphorothioate-containing RNAs were generated by transcription of template DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) (NTP alpha S) and T7 RNA polymerase. The substitution of mRNA by phosphorothioate increased the efficiency of protein synthesis by stabilizing the mRNAs in prokaryotic cell-free translation systems. The substituted mRNAs were also shown to be applicable to the continuous cell-free translation system developed by Spirin and coworkers.
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PMID:Cell-free translation system using phosphorothioate-containing mRNA. 172 4

A kinetic assay has been developed to measure the strength of natural T7 promoters. By determining the rate of appearance of initiation products in the presence of constant concentrations of T7 RNA polymerase, an incomplete mixture of ribonucleoside triphosphates, and increasing promoter concentrations, a maximum rate of product formation (Vmax) and a promoter concentration giving half of the maximal activity ([P]Vmax/2) can be determined for any cloned T7 promoter. On supercoiled plasmids, it was found that the [P]Vmax/2 measured for the six promoters phi 1.1B, phi 1.3, phi 3.8, phi 6.5, phi 10, and phi 13 ranged from 3.4 +/- 1.1 to 12.0 +/- 2.4 nM while the Vmax values showed no significant trends. On plasmids that had been linearized by cleavage at a single site with a restriction endonuclease, the cloned T7 promoters assayed fell into two broad classes that appear to be characterized by the T7 class II and III promoters. Generally, the class II promoters required higher promoter concentrations to produce half of the maximum rates of initiation ([P]Vmax/2 values) than the class III promoters. The [P]Vmax/2 values for the class II promoters ranged from 20 +/- 2.7 to 23 +/- 3.6 nM, while the [P]Vmax/2 values for the class III promoters phi 10 and phi 13 were 13 +/- 1.6 nM and 7.8 +/- 1.4 nM. The one exception is the class III promoter phi 6.5 whose [P] Vmax/2 (17 +/- 5 nM) falls between the [P]Vmax/2 values of the class II promoters and the strong class III promoters. The Vmax values measured on linear templates are variable, but it appears that phi 10 is more active than the other five promoters.
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PMID:Initiation of transcription by T7 RNA polymerase as its natural promoters. 173 60

Many RNA polymerases stall and/or prematurely terminate transcription nearby the early and late promoters of polyomavirus in vivo during the late phase of productive infection. In this paper we analyzed the RNAs made when these promoter-proximal RNA polymerases were allowed to elongate their nascent chains in vitro on viral transcription complexes isolated from infected cells. RNA was labeled in the presence of a high specific activity of one [alpha-32P]-ribonucleoside triphosphate (rNTP) (less than 1 microM final concentration) and saturating concentrations of the other three rNTPs. Under these conditions, promoter-proximal RNAs of discrete sizes were produced. We show that these discrete RNA species are produced by pausing of RNA polymerase II due to limiting concentrations of one of the rNTPs, and that the positions of the pause sites depend on which rNTP is limiting. This pausing does not result in release of the RNA polymerase or the nascent RNA chain from the transcription complex, as these chains can be further extended when high concentrations of all four rNTPs are supplied. Our results conflict with the interpretations of other investigators who suggest that formation of discrete RNA products, under similar in vitro conditions, reflects authentic termination by RNA polymerase II.
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PMID:RNA polymerase II pauses in vitro, but does not terminate, at discrete sites in promoter-proximal regions on polyomavirus transcription complexes. 185 Sep 13

We have used a self-cleaving RNA molecule (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates. When the nascent transcript attains the minimum length required for the "hammerhead" domain of the transcript to fully emerge from the ternary complex, the "hammerhead" structure forms and self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to that generated by using a noncleaving control template. We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.
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PMID:RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay. 193 16


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