共查询到20条相似文献,搜索用时 15 毫秒
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Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. 总被引:11,自引:0,他引:11
E Scherzinger E Lanka G Morelli D Seiffert A Yuki 《European journal of biochemistry》1977,72(3):543-558
The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo. 相似文献
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Purified cores of vesicular stomatitis virus contain an enzymatic activity that converts GDP, UDP, and CDP into their corresponding triphosphates using ATP as the phosphate donor. Thus, the virion-associated RNA polymerase can synthesize mRNA normally in vitro even when one of the ribonucleoside triphosphates is replaced by its corresponding diphosphate. RNA synthesis does not proceed if ATP is replaced by ADP. Similarly RNA synthesis is impaired if CDP and UDP are present in the same reaction. The role of the nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6) in vesicular stomatitis virus mRNA synthesis in vitro is discussed. 相似文献
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Characterization of a temperature-sensitive defect of enterovirus 70: effect of elevated temperature on in vitro transcription 总被引:1,自引:1,他引:0
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A crude replication complex prepared from enterovirus 70-infected cells was used to study the temperature-sensitive characteristic of the virus. The complex showed a temperature sensitivity in the in vitro incorporation of radiolabeled ribonucleoside triphosphate. The endonuclease itself did not account for the restricted RNA synthesis at the nonpermissive temperature. Analyses of the in vitro products by both gel electrophoresis and sucrose density gradient centrifugation showed that the complex synthesized three types of viral RNA only when incubated for a short period of time at the nonpermissive temperature. When the replication complex was treated with a detergent (deoxycholic acid), incorporation of ribonucleoside triphosphate into RNA at the permissive temperature was reduced to the level of that at the nonpermissive temperature. In addition, the in vitro RNA synthesis by the enterovirus 70 replication complex at the permissive temperature required a higher concentration of ATP than of other ribonucleoside triphosphates, whereas such a preference for ATP was not found in the reaction at the nonpermissive temperature. The results indicate that the initiation step of RNA synthesis by the complex is blocked at the nonpermissive temperature. The possible implications of these findings are discussed. 相似文献
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W L Staudenbauer 《European journal of biochemistry》1975,58(2):303-313
Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D. 相似文献
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A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol. 相似文献
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《Gene Analysis Techniques》1986,3(4):59-66
The 3′-deoxy and 3′ -0-methyl analogs of the standard ribonucleoside triphosphates were found to act as base-specific chain terminators of RNA synthesis mediated by the T3 RNA polymerase. Because this enzyme initiates RNA synthesis at a unique site within its promoter sequence, all RNA chains initiated at a cloned promoter have a common 5′ terminus. The specifically terminated products that are made in the presence of the analogs may be resolved by gel electrophoresis, permitting the determination of the nucleotide sequence of the DNA template from which the RNA was transcribed. These findings demonstrate that the T3 RNA polymerase can provide the basis of a useful method for determining the sequence of double-stranded DNA templates. 相似文献
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Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA. 相似文献
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Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.
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Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates. 相似文献
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RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES 总被引:1,自引:0,他引:1
Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state. 相似文献
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Pre-4S RNA made in isolated HeLa cell nuclei terminates with U 总被引:3,自引:0,他引:3
Isolated HeLa cell nuclei are capable of synthesizing a precursor to 4S RNA. The 3′ terminus of the molecule made in vitro can be determined by four separate incubations with the four iritiated ribonucleoside triphosphates. The pre-4S was found to terminate specifically with uridine and be uniformly labeled. These facts are discussed in relation to specific termination of RNA molecules and possible reinitiation of RNA synthesis in isolated nuclei. 相似文献
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Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis 总被引:13,自引:2,他引:11
The ribonucleotide and deoxyribonucleotide contents of eggs and oocytes of Xenopus laevis were measured. Eggs contained most deoxyribonucleotide in the form of triphosphates. dCTP, dTTP, dATP and dGTP were present in similar amounts. The egg contained sufficient deoxynucleotide triphosphate to make approximately 2500 nuclei. Oocytes contained less pyrimidine deoxyribonucleoside triphosphates than did eggs, and purine deoxyribonucleoside triphosphates were not detected. These differences may be correlated with the ability of eggs to induce nuclear DNA synthesis, a property not shown by oocytes. Both oocytes and eggs seem to contain non-phosphorylated, alpha-unsubstituted aldehydes, which may be deoxyribose derivatives. Eggs and oocytes contain similar amounts of ribonucleoside triphosphates. The low rate of RNA synthesis found in eggs, but not in oocytes, is therefore not caused by simple precursor control. 相似文献
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We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E. coli replication fork. Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces. All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used. However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction. A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture. Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available. 相似文献
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Shonen Yoshida Rika Suzuki Shigeo Masaki Osamu Koiwai 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(3):348-357
Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA. 相似文献
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G Meneguzzi P F Pignatti G Milanesi 《Biochemical and biophysical research communications》1976,72(2):626-633
Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent 32PPi exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase. 相似文献
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G Banfalvi V Slezarikova M Sedliakova F Antoni 《European journal of biochemistry》1987,162(2):305-309
DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%. 相似文献
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William S. Robinson 《Journal of virology》1971,8(1):81-86
After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA. 相似文献
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