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)

rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.
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PMID:Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator. 152 50

New spin-labeled analogs of nucleoside triphosphates, 8-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenosine 5'-triphosphate ((8-AmTEMPO)ATP) and 5-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)uridine 5'-triphosphate ((5-AmTEMPO)UTP), with the probe 4-amino(2,2,6,6-tetramethylpiperidine-N-oxyl) (4-AmTEMPO) attached to C-8 of ATP and C-5 of UTP via a secondary amine bond, were synthesized in 50 and 40% yield, respectively. These analogs showed a single spot by thin layer chromatographic analysis. The absorption spectra of (8-Am-TEMPO)ATP and (5-AmTEMPO)UTP exhibit maxima at 310 and 265 nm, respectively; their X-band EPR spectra have a typical three-line pattern with lines at 3,221, 3,239, and 3,257 Gauss. The intensity ratios for mid to high field lines of the EPR derivative lines were found to be 1.03 +/- 0.02, 1.08 +/- 0.04, and 1.15 +/- 0.07 for 4-AmTEMPO, (8-AmTEMPO)ATP, and (5-AmTEMPO)UTP, respectively. The immobilization of 4-AmTEMPO bound to C-8 of ATP or bound to C-5 of UTP was observed to be 5 and 11%, respectively, as compared with free 4-AmTEMPO. The initial velocity (s-1) of [3H]UMP incorporation into RNA in the presence of [3H]UTP, CTP, GTP, and (8-AmTEMPO)ATP or ATP was measured. The percent incorporation of (8-AmTEMPO)ATP into RNA product by Escherichia coli RNA polymerase using various DNA templates is 68, 66, and 61% for pAR1435 (plasmid containing A1 promoter from T7 DNA), calf thymus DNA, and poly(dA-dT) respectively, as compared with ATP incorporation. The polymerase-catalyzed reaction of (8-AmTEMPO)ATP with (3'-OCH3)UTP yielded 5'-triphosphate delta-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenylyl (3'-5')3'-methoxy uridine in the presence of poly(dA-dT). The structure of this spin-labeled dinucleotide was identified by paper chromatographic analysis of the products of phosphodiesterase digestion. These analogs also can be used for the study by EPR spectroscopy of the dynamics of gene transcription catalyzed by RNA polymerases or of other nucleotide-utilizing enzymes.
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PMID:Spin-labeled nucleotide substrates for DNA-dependent RNA polymerase from Escherichia coli. 165 31

Tagetitoxin, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. tagetis, inhibits RNA synthesis directed by chloroplast RNA polymerase. In isolated chloroplasts, tagetitoxin quickly and specifically reduced the incorporation of [3H]uridine into RNA. When it was added to transcriptionally active chloroplast protein extracts, the toxin directly inhibited incorporation of [32P]UTP into RNA. In addition, tagetitoxin inhibited in vitro RNA synthesis directed by the RNA polymerase from Escherichia coli. In vitro transcription reactions directed by chloroplast RNA polymerase or E. coli RNA polymerase are inhibited at tagetitoxin concentrations less than 1 microM. Nuclear RNA polymerase II purified from wheat germ was only affected at tagetitoxin concentrations greater than 100 microM during in vitro transcription. Tagetitoxin concentrations as high as 1 mM did not affect in vitro transcription reactions directed by RNA polymerase from bacteriophage T7 or SP6.
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PMID:Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli. 168 34

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

Nuclei isolated from senescent (22-26 months) female Wistar rat liver show a decreased RNA synthesis compared to nuclei from adult (12 months) liver. Kinetic analyses demonstrate a decreased rate (25-42%) and maximal incorporation (41-54%) of labeled uridine triphosphate into both rRNA and mRNA in senescent nuclei. Chromatin-bound RNA polymerase activity is decreased by 36%, whereas free RNA polymerase activity, i.e., not bound to chromatin, is increased by 41% in senescent nuclei, but the total bound plus free activity is the same in senescent and adult nuclei. Isolated senescent chromatin shows reduced transcriptional capacity and requires a higher temperature to initiate melting. A decreased ability of chromatin to bind RNA polymerases appears to underlie the observed decreased RNA synthesis in senescent liver nuclei.
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PMID:RNA synthesis by nuclei and chromatin isolated from adult and senescent Wistar rat liver. 169 84

The polyamines of pilose antler (PASPA) consist of putrescine (PU, 70.9%), spermidine (SPD, 26.3%) and spermine (SP, 2.8%). The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver tissue were increased when PASPA was given orally to mice at the dose of 30 mg/kg for 4 successive days. The incorporations of [3H] leucine into liver protein and [3H] uridine into the cytosolic and nuclear RNA were also increased by treatment with PU (21 mg/kg). In addition, the RNA polymerase activity in the solubilized liver nuclear fraction of PU (21 mg/kg)-treated mice was increased. SPD only promoted the synthesis of protein in mouse liver tissue at the dose of 8 mg/kg. However, SP showed no effect on the synthesis of protein and RNA polymerase activity under the used dose (1 mg/kg). The results suggest that PASPA is the main active substance responsible for the promotion of the synthesis of protein and RNA in mouse liver.
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PMID:[Influence of the active compounds isolated from pilose antler on syntheses of protein and RNA in mouse liver]. 170 79

The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver were increased when PASPA was given to mice at a dose of 30 mg/kg for 4 successive days. The RNA polymerase activity, especially the RNA polymerase II activity in the solubilized liver nuclear fraction of PASPA-treated mice was also increased. In vitro experiment demonstrated that PASPA increased the RNA polymerase activity significantly in mouse liver nuclei at a concentration of 1 microgram/ml. These results suggest that the enhancement of RNA polymerase activities, particularly RNA polymerase II activity, induced by PASPA treatment is responsible for the increase in synthesis of protein and RNA in mouse liver tissue.
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PMID:[Effect of polyamines isolated from pilose antler (PASPA) on RNA polymerase activities in mouse liver]. 170 30

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.
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PMID:Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes. 171 Sep 32

The kinetics of formation of abortive initiation products during transcription of a synthetic template (encoding the transcript GAUGGC) by T7 RNA polymerase have been determined. This study revealed that while total RNA was formed in the reaction as expected, the levels of the dinucleoside tetraphosphate guanylyl-3',5'-adenosine-5'-triphosphate (pppGpA) and trinucleoside pentaphosphate guanylyl-3',5'-adenosine-3',5'-uridine-5'-triphosphate (pppGpApU) formed by premature termination of transcription reached a maximum after 10 min, and then decreased. Transcription of the same template, in the presence of either [gamma-32P]GTP and ATP, or GTP and [alpha-32P]ATP, gave the 32P-labeled dinucleotides *pppGpA and pppG*pA. Incorporation of each of these substrates into longer RNA transcripts in the same enzyme-template system was demonstrated. The incorporation was shown to require the presence of template in the reaction mixture. The requirement for base complementarity restricts the position of incorporation to that of initiating (5') nucleotide. Transcription of a second template, which encodes an RNA transcript having the partial sequence GpA at two internal positions, in the presence of each of the labeled dinucleoside tetraphosphates, failed to bring about the synthesis of significant yields of any longer radiolabeled transcripts. It is concluded that dinucleoside tetraphosphate (and perhaps trinucleoside pentaphosphate) can function as initiating nucleotides when complementary to the nucleotide sequence at promoter regions. However, a dinucleotide is not used as substrate for subsequent chain elongation in T7 RNA polymerase catalyzed transcription reactions.
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PMID:Abortive products as initiating nucleotides during transcription by T7 RNA polymerase. 171 17

Autoradiographic and immunofluorescent techniques have been used to analyse the relationship between puffing, transcription and occurrence of DNA/RNA hybrids in D melanogaster salivary gland chromosome 2L. Experiments of 3H-uridine incorporation have indicated that similar rates of RNA synthesis are observable in well developed puffs as well as in some diffuse bands and interbands. On the other hand, puffs of similar size incorporate 3H-uridine at quite different rates. The presence of RNA polymerase II seems to follow a coincident pattern with that of 3H-uridine incorporation. Our results indicate that the rate of transcription does not determine either the formation of a puff or its potential size. Instead, we have found a positive correlation between the amount of DNA/RNA hybrids and puff size, independently of the transcription rates. Transient accumulation of transcribed RNAs in their chromosomal compartment could therefore play a relevant role in the determination of puff size.
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PMID:Comparative analysis of four parameters involved in puffing activity along chromosome arm 2L of D melanogaster. 172 21


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