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 nuclear DNA of HeLa cells can now be isolated unbroken and supercoiled. Using DNA gyrase and the untwisting enzyme, we have prepared an allomorphic series of templates derived from this nuclear DNA, and also from the circular DNA of the bacterial virus, PM2. We have then transcribed these templates using 2 different RNA polymerases--from wheat germ and Escherichia coli. Relaxed DNA is transcribed slowly by both polymerases. Supertwisting the naturally-supercoiled templates with gyrase slightly inhibits transcription by the bacterial polymerase but stimulates dramatically transcription by RNA polymerase II from wheat germ.
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PMID:DNA gyrase stimulates transcription. 625 26

The parameters governing the activity of the cloned T4 gene 23, which codes for the major T4 head protein, were analyzed. Suppressor-negative bacteria carrying wild-type T4 gene 23 cloned into plasmid pCR1 or pBR322 were infected with T4 gene 23 amber phage also carrying mutations in the following genes: (i) denA and denB (to prevent breakdown of plasmid DNA after infection) and (ii) denA, denB, and, in addition, 56 (to generate newly replicated DNA containing dCMP) and alc/unf (because mutations in this last gene allow late genes to be expressed in cytosine-containing T4 DNA). Bacteria infected with these phage were labeled with (14)C-amino acids at various times after infection, and the labeled proteins were separated by one-dimensional gel electrophoresis so that the synthesis of plasmid-coded gp23 could be compared with the synthesis of other, chromosome-coded T4 late proteins. We analyzed the effects of additional mutations that inactivate DNA replication proteins (genes 32 and 43), an RNA polymerase-binding protein (gene 55), type II topoisomerase (gene 52), and an exonuclease function involved in recombination (gene 46) on the synthesis of plasmid-coded gp23 in relation to chromosome-coded T4 late proteins. In the denA:denB:56:alc/unf genetic background, the phage chromosome-borne late genes followed the same regulatory rules (with respect to DNA replication and gp55 action) as in the denA:denB genetic background. The plasmid-carried gene 23 was also under gp55 control, but was less sensitive than the chromosomal late genes to perturbations of DNA replication. Synthesis of plasmid-coded gp23 was greatly inhibited when both the type II T4 topoisomerase and the host's DNA gyrase are inactivated. Synthesis of gp23 was also substantially affected by a mutation in gene 46, but less strongly than in the denA:denB genetic background. These observations are interpreted as follows. The plasmid-borne T4 gene 23 is primarily expressed from a late promoter. Expression of gene 23 from this late promoter responds to an activation event which involves some structural alteration of DNA. In these respects, the requirements for expressing the plasmid-borne gene 23 and chromosomal late genes are very similar (although in the denA:denB:56:alc/unf genetic background, there are significant quantitative differences). For the plasmid-borne gene 23, activation involves the T4 gp46, a protein which is required for DNA recombination. However, for the reasons presented in the accompanying paper (Jacobs et al., J. Virol. 39:31-45, 1981), we conclude that the activation of gene 23 does not require a complete breakage-reunion event which transposes that gene to a later promoter on the phage chromosome. Ways in which gp46 may actually be involved in late promoter activation on the plasmid are discussed.
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PMID:Regulation of expression of cloned bacteriophage T4 late gene 23. 626 20

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.
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PMID:Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication. 627 38

A temperature-sensitive mutant of E. coli with a defective DNA gyrase B subunit has been obtained. The mutation is expressed in the thermolability of DNA gyrase in vitro and in DNA relaxation in vivo. DNA replication in the mutant does not stop under non-permissive conditions; its rate gradually falls by a factor of 2 to 3. The transcription rate also drops by a factor of 2 to 3, but before replication. Small concentrations of rifampicin, an inhibitor of bacterial RNA polymerase, make for a partial survival of the mutant cells under non-permissive conditions. The results suggest the conclusion that DNA supercoiling is mainly required to ensure the optimum transcription level in the cell.
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PMID:DNA replication and transcription in a temperature-sensitive mutant of E. coli with a defective DNA gyrase B subunit. 629 83

A temperature sensitive mutant affecting the B-subunit of DNA gyrase was isolated. The mutation leads to the thermolability of DNA gyrase in vitro and to the plasmid DNA relaxation in vitro. The immediate stop of the increase in mutant culture titre has been observed under nonpermissive conditions. However, DNA synthesis does not cease, though its efficiency is reduced by a factor of three. The transcription rate is also reduced two - three times, but more quickly than the rate of replication. This would mean that the transcription apparatus is the first to react to the change of DNA supercoiling in the cell. This suggestion is supported by the facts that small amounts of rifampicin increase the viability of ts mutant cells under nonpermissive conditions and also by the previously obtained results concerning the change in sensitivity of RNA polymerase mutants to DNA gyrase inhibitors.
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PMID:[Temperature-sensitive mutant Escherichia coli for the B-subunit of DNA-gyrase. The effect on replication and transcription]. 630 2

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.
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PMID:Evidence for participation of a multiprotein complex in yeast DNA replication in vitro. 637 67

To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in phi29 DNA replication, extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex. Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex, suggesting that these proteins are not involved in the initiation of phi29 DNA replication. ddATP or aphidicolin, inhibitors of DNA chain elongation, had also no effect on the formation of the initiation complex.
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PMID:Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex. 640 61

A specific inhibitor of DNA gyrase, coumermycin A1, inhibits in vivo the interaction of bacteriophage lambda DNA and DNA of rifd transducing phages (lambda rifd47 and lambda att80 rifd35) carrying genes rpoB and rpoC of RNA polymerase. Besides, coumermycin A1 inhibits recA-dependent recombination between the bacterial region of rifd phages and a homologous region of Escherichia coli chromosome. The integration of transdusing phage DNA into the chromosome of recA host is significantly more sensitive to the inhibitory effect of coumermycin A1 than the two other processes (by one -- three orders or magnitude). Another inhibitor of DNA gyrase, nalidixic acid, does not depress the integration of both lambda and rifd phages DNA. The differential effect of coumermycin A1 on the integration of lambda and rifd phages correlates with our concept of the int-idependent mechanism of rifd phage DNA integration in recA bacteria. According to preliminary results, coumermycin A1, in contrast to its action on integration, does not inhibit the exclusion of lambda prophage from E. coli chromosome, while nalidixic acid produces a slight inhibitory effect.
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PMID:[Effect of coumermycin A1 and nalidixic acid on lambda phage integration and transducing lambdoid phages containing RNA-polymerase genes]. 645 Jul 9

Exogenous lambda dv DNA was replicated in extracts prepared from E. coli cells carrying plasmids with inducible lambda O and /or P genes. Extracts from cells carrying only one of the two lambda replication functions complement each other in this reaction. The reaction further requires ribonucleotide triphosphates, an ATP regenerating system, DNA gyrase and RNA polymerase functions. Density labelling of the superhelical reaction products results in hybrid density indicating that one complete round of replication has taken place in vitro.
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PMID:Replication of lambda dv DNA in vitro. 646 46

Replication of lambda plasmid DNA is inhibited in amino acid-starved wild type Escherichia coli cells (i.e., during the stringent response), whereas it proceeds for several hours in relA mutants (i.e., during the relaxed response). It was demonstrated previously that ppGpp-mediated inhibition of transcription starting from the pR promoter is responsible for inhibition of lambda plasmid replication; RNA polymerase function is indispensable for replication of lambda plasmid DNA during the relaxed response. The replication is carried out by the heritable replication complex containing the lambda O protein which is protected from proteases by other elements of this complex. Here we demonstrate that the replication is dependent on DnaG (primase) function. Thus, in amino acid-starved cells, lambda plasmid replication requires RNA polymerase function only for transcriptional activation of ori lambda. We also present evidences that the replication is dependent on the function of DNA gyrase. On the basis of these findings and other recent reports, we demonstrate a model of the regulation of lambda plasmid replication driven by the inherited replication complex. It seems that transcriptional activation of ori lambda indirectly triggers the initiation of lambda plasmid DNA replication at least during the relaxed response.
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PMID:Transcriptional activation of ori lambda regulates lambda plasmid replication in amino acid-starved Escherichia coli cells. 757 72


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