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)

The subunit composition of Escherichia coli RNA polymerase during the transcription in vitro of bacteriophage T7DNA was analysed at several steps of RNA synthesis. RNA-polymerase . DNA complexes were sedimented through a glycerol gradient and the RNA polymerase subunits present in each fraction of the gradient were separated by dodecylsulfate-polyacrylamide gel electrophoresis and quantified colorimetrically on the gels. RNA polymerase selectively bound to T7 DNA in the absence of nucleoside triphosphates has the same subunit composition as free RNA polymerase holoenzyme (beta'betaalpha2) omicron. Addition of the nucleoside triphosphate combinations ATP, GTP, UTP or ATP, CTP, UPT, or GTP, CTP UTP to the binding reaction does not alter the subunit composition of RNA polymerase holenzyme bound to DNA. In contrast, in the presence of ATP, GTP and CTP up to 3 pmol of omicron-subunit are released from a complex containing RNA polymerase and 1 pmol of T7 DNA. In the presence of the four nucleoside triphosphates about 90% of the RNA polymerase associated with DNA and nascent RNA has the subunit composition of RNA polymerase core enzyme (bet'betaalpha2). The omicron-subunit is released from the complex and is recovered near the top of the gradient. The transition from the binding complex to the elongation complex and the incorporation of gamma32P-labeled ATP and GTP at the 5' end of RNA molecules were followed in parallel. In the purified elongation complex about 1 pmol of ATP or GTP is incorporated into RNA per pmol RNA polymerase core enzyme engaged in RNA synthesis.
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PMID:Subunit composition of Escherichia coli RNA polymerase during transcription in vitro. 77 24

EcoRI fragments A, B and C produced from linear phi29 DNA, but not D or E fragments, are transcribed by purified Bacillus subtilis RNA polymerase. The transcription of fragments A and C is initiated preferentially with GTP and to a lesser extent with ATP; the reverse happens in the case of fragment B. The dinucleotides GpU and GpA respectively, compete specifically with the incorporation of [gamma-32P]GTP directed by fragments A and C. The RNA synthesized in vitro by purified B. subtilis RNA polymerase is highly asymmetric. Most of the RNA synthesis directed by fragments A and C is early RNA. However, most of the RNA produced by fragment B is anti-late-RNA. Addition of crude extracts inhibit the transcription of fragment B but not that of fragments A and C.
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PMID:Transcription in vitro of phi29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase. 82 46

The effect of the dye aurintricarboxylic acid (ATA) on RNA polymerases [EC 2.7.7.6] solubilized from rat liver was studied. Complete inhibition of RNA synthesis in vitro was observed when 3-5 microng/ml of ATA was added to the reaction mixture at time 0, while 40-70 microng/ml of a rifampicin derivative, AF/013, was required to produce the same extent of inhibition. RNA formation, however, continued at a rate of one-half that of the control when ATA was given after the onset of RNA synthesis in a dose capable of completely blocking RNA formation if administered at time 0. ATA was found to interact with RNA polymerizing enzyme itself and competed specifically with the binding of RNA polymerase to template DNA. Preincubation of the enzyme with DNA at 37 degrees before adding dye made the DNA-enzyme complex partly resistant to the drug. RNA-synthesizing activity resistant to ATA increased when nucleoside triphosphates, especially GTP, were added to the preincubation mixture in the presence of Mn2+. However, ATA only slightly affected RNA synthesis in nuclei isolated from rat liver.
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PMID:The effect of aurintricarboxylic acid on RNA polymerase from rat liver. 84 32

Nuclei from seminal vesicle epithelium of adult guinea pigs were isolated in hypertonic sucrose solution. The incorporation of [3H]UTP by the isolated nuclei into acid-precipitable products was studied. Incorporation required ATP, GTP, CTP, UTP, and Mg+2. It was inhibited by addition of actinomycin D, deoxyribonuclease, or pyrophosphate to the reaction mixture. Thus, incorporation of [3H]UTP by isolated nuclei had the same characteristics that have been demonstrated for the reactions catalyzed by nuclear RNA polymerases. Using alpha-amanitin as a metabolic tool, we established concentrations of (NH4)2SO4. Mg+2, and nucleotides that give maximum assayable activities of nuclear RNA polymerases I and II. When the activities of polymerases I and II were measured in isolated seminal vesicle nuclei of guinea pigs that had been castrated 4 days earlier, a marked decrease in activities was found relative to control values (nuclei from intact animals). No further decrease was found 8 days after castration. Diminished accessibility to the nuclear DNA template and a decrease in the concentration of RNA polymerase molecules seemed to be responsible for the observed effects of castration on activities of RNA polymerases. An increase in ribonuclease activity did not seem to be responsible for the effects of castration. Activities of the enzymes did not change 2, 3, or 4 hours after intraperitoneal injection (2 mg/kg body weight) of each of five different androgens. Similarly, a single intraperitoneal injection of testosterone did not restore enzyme activity of polymerade I or II at any time during the first 24-hour period after hormone administration.
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PMID:RNA polymerase activities in isolated nuclei of guinea pig seminal vesicle epithelium: influence of castration and androgen administration. 90 9

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA. Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to alpha-amanitin. In isolated nuclei, both RNA species are labeled with beta-32P-labeled GTP, which suggests that they are initiated at separate promotor sites.
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PMID:A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2. 95 88

Using RNA polymerase purified from Escherichia coli, DNA isolated from the bacteriophage T4, and a bacterial supernatant fraction containing the necessary processing enzymes, a set of transfer RNAs can be formed in vitro. To characterize the site or sites of initiation of this tRNA transcription, rifampicin-resistant complexes of RNA polymerase, DNA, and either ATP (UTP and CTP) or GTP (UTP and CTP) were formed, and tRNA was transcribed from these stabilized sites. It is concluded that transcription of the entire set is initiated by ATP. To study the transcription of the tRNAs, the time sequence of the appearance of individual species was determined during synchronous transcription of a preformed RNA polymerase-DNA complex. The appearance of three RNA species is found to be consistent with the sequential transcription of a large polycistronic cluster; the order and distances, inferred from the times of transcription, are as required by the existing gene map. It is concluded that the initiation of tRNA transcription can occur, without accessory factors, with the insertion of ATP at a single or a few closely spaced sites, and that the tRNAs encoded by the bacteriophage T4 are present in a single operon.
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PMID:Initiation and transcription of a set of transfer ribonucleic acid genes in vitro. 108 54

These experiments investigate two aspects of RNA synthesis in Escherichia coli ML30 during the transition from a relatively slow rate of growth to a more rapid one: (1) the number of growing RNA molecules per cell, and (2) the average time required for addition of a nucleotide onto a growing RNA chain. Cells were grown at 30 degrees C in a glucose-minimal salts medium and shifted-up by the addition of Casamino acids. Measurements were made of the rates of incorporation over short intervals (e.g. 5,8,12, and 16 s) of [3-H]guanine into the internal and 3'-terminal nucleotides of RNA. After correction for the specific activities of the intracellular GTP pools, and for the rate of [3-H]guanine accumulation at the 3'-terminus of non-growing RNA, the rates of chain elongation were calculated. It was found that cells growing at a rate of 0.9 generations/h contain approx. 4800 RNA molecules, growing at a rate of 28 nucleotides/s per chain. Cells growing exponentially at the postshift-up rate (1.2 generations/h) contain 7000 RNA molecules per unit equivalent cell mass, which are growing at a rate of 32 nucleotides/s per molecule. Three min after shift-up, cells contain the same number or slightly fewer (10%) growing RNA molecules than cells prior to shift-up, 4300, and these are being elongated at a rate of about 32 nucleotides per s. The results are consistent with the view that in the range of growth rates studied, the total rate of RNA synthesis is regulated through a limitation in the number of functioning RNA polymerase molecules, each working at a relatively constant, presumably maximal, average rate.
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PMID:Regulation of RNA synthesis in Escherichia coli during a shift-up transition. 109 70

The synthetic DNA alternating copolymers poly dAT-dAT and dABU-dABU have been transcribed with E. coli RNA polymerase to measure the level of BrdU-induced misincorporation of guanine during transcription. GTP is found to be misincorporated into both copolymers at a frequency of 1 per 1000-2000 nucleotides polymerized. Using alpha-32P-GTP, the nearest neighbors to GMP are found to be UMP (approximately 63%), GMP (approximately 25%) and AMP (approximately 17%), with no apparent difference between the two templates. These results suggest that BrdU-substitution in DNA does not necessarily increase the potential for base mispairing during transcription, and hence, promote the production of faulty RNA molecules.
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PMID:Misincorporation of (TP during transcription of poly dAT-dAT and poly dABU-dABU. 110 Dec 26

Circular dichroic spectra of T7 RNA polymerase show minima at 222 nm ([theta]m=-7.9 X 10(3) deg cm2/dmol) and 208 nm ([theta]m =-7.55 X 10(3) deg cm2/dmol) and a maximum at 193 nm ([theta]m = 1.2 X 10(4) deg cm2/dmol). The small mean residue ellipticity above 200 nm indicates that the secondary structure contains approximately 12% alpha helix. The secondary structure is unaltered by high salt, glycerol, -SH reagents, nitration of tyrosyl residues, and chelating agents. Binding of the native enzyme to [32P]T7 DNA has been measured by the retention of the protein-[32P]DNA complexes on nitrocellulose filters. At 37degrees T7 RNA polymerase binds to its promoters in the absence of NTP's. Binding and catalytic activity are both abolished at 0degree. Binding of the initiating [gamma-32P]GTP can also be detected by the filter binding assay. Native T7 RNA polymerase is inactivated by reaction with 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or 1 mol of [14C]iodoacetamide. The latter reaction is blocked by Nbs2 suggesting that a single -SH group is required for activity. Alkylation of the -SH group does not alter binding of the enzyme to the DNA template, but modifies the binding of GTP to the enzyme. Nitration of approximately4 surface tyrosyl residues of the protein prevents binding to T7 DNA. The restriction endonuclease, Hpa II, cuts T7 DNA into approximately40 fragments and reduces total RNA synthesis by T7 RNA polymerase by 70%. Fragmentation of the DNA template by Hpa II does not alter the rate of RNA chain initiation by T7 polymerase, and restriction fragments accounting for approximately25% of the T7 DNA still bind tightly to the enzyme. Thus the T7 RNA polymerase promoters remain intact on the restriction fragments. Gel electrophoresis of the transcription products, using restriction fragments as templates, show that of the seven in vitro transcripts produced by T7 RNA polymerase from whole T7 DNA, only the smallest (representing the last 1.5% of the genome) is transcribed from Hpa II fragments. The remaining transcripts are replaced by six new and much shorter mRNA's. The DNA fragments containing the promoters for these mRNA's have been removed from the fragment mix by binding them to the enzyme and retaining the complexes on nitrocellulose filters.
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PMID:T7 RNA polymerase: conformation, functional groups, and promotor binding. 110 55

The kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed. Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate. In this reaction RNA polymerase serves as a reactant rather than acting catalytically. The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used. Kinetic equations for an ordered two-substrate reaction are derived. These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin. Equations for the latter instance are solved exactly with recourse to the steady state approximation. It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis. This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase. It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule. The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions. Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain.
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PMID:Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. 110 16


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