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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)

DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30 degrees C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E.coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few "eye"-shaped structures resembling the early replicative intermediates normally observed in vivo.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins. 5 68

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images
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PMID:Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein. 33 93

H1 factor is a heat-stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of lambda templates by E. coli DNA-dependent RNA polymerase. The subunit molecular weight of this factor has been re-estimated and found to be 15500 +/- 1000 in the presence of 2-mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15500-Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000-Mr subunit is labeled by 4-chloro-7-nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated. H1 factor behaves as a DNA-binding protein. We have detected binding to DNA by two independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of lambda plac DNA as well as of wild-type lambda DNA has been found to be non-cooperative. Saturation is reached when 300 +/- 30 molecules of dimeric factor are bound per lambda molecule, the average dissociation constant of the complex being 10nM. The dissociation time of the H1.DNA complex is less than 5 s at 37 degrees C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.
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PMID:Physico-chemical properties of a DNA binding protein: Escherichia coli factor H1. 33 3

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase.
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PMID:Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. 34 Apr 57

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

A Wilms' tumor susceptibility gene (WT1) localized to 11p13 was recently isolated and shown to be altered in some sporadic Wilms' tumors. This gene encodes a DNA-binding protein with four zinc fingers (ZFs) in the carboxy-terminal region and a glutamine/proline (Gln/Pro)-rich domain near the 5' end. Two alternative splice sites were described, splice I in the Gln/Pro-rich domain (51 bp) and splice II between ZFs 3 and 4 (9 bp). Using RNA polymerase chain reaction (PCR) we show that Wilms' tumors contain all four possible transcripts, which are also identified in normal adult and embryonic kidney cells. The transcripts containing the 9-bp ZF insert were always predominant in tumors and normal cells. The presence of all four WT1 transcripts in tumors and expressing tissues suggests that each encoded protein isoform has an important role for the function of the WT1 gene.
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PMID:RNA polymerase chain reaction detects different levels of four alternatively spliced WT1 transcripts in Wilms' tumors. 132 Feb 46

BC-1 RNA is a brain-specific small RNA transcript of identifier sequences present in the somas and dendrites of neurons. We recently reported that the RNA is complexed with a protein(s) to form a 10 S ribonucleoprotein particle (Kobayashi, S., Goto, S., and Anzai, K. (1991) J. Biol. Chem. 266, 4726-4730). We demonstrate here that this 10 S BC-1 ribonucleoprotein particle contains a DNA-binding protein(s) (Bp-1 protein) capable of interacting with a region between split promoter sequences for RNA polymerase III within the identifier sequences. The region has short inverted repeats: a perfect octanucleotide repeat (GCGCTTGCCTAGCAAGCGC) and an imperfect heptanucleotide repeat (GCCTAGCAAGCGCAAGGC), each of which contains a GCAAG/CTTGC motif. We also demonstrate that the binding of this protein either to the array of pentamer motifs or to BC-1 RNA is mutually exclusive. The molecular masses of photo-cross-linking adducts of Bp-1 protein to a 32P-labeled GCAAG/CTTGC motif-specific probe were estimated to be about 31 and 36 kDa, indicating that two species of Bp-1 proteins may be present in the brain.
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PMID:The 10 S BC-1 ribonucleoprotein particle contains identifier sequence-binding proteins that interact with an array of GCAAG/CTTGC motifs between split promoter sequences for RNA polymerase III. 138 53

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.
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PMID:Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. 145 53

Sporulation of Bacillus subtilis involves the differentiation of two cell types, the mother cell and the forespore. Two key regulators of mother-cell gene expression are SpoIIID, a DNA-binding protein that activates or represses transcription of many different genes, and sigma K, a subunit of RNA polymerase that directs the enzyme to transcribe genes encoding proteins that form the spore coat. Previous studies showed that SpoIIID is needed to produce sigma K, but suggested that SpoIIID represses sigma K-directed transcription of genes encoding spore coat proteins. Here we show that a feedback loop connects the levels of sigma K and SpoIIID, such that production of sigma K leads to a decrease in the level of SpoIIID. The existence of the feedback loop was demonstrated by using antibodies prepared against SpoIIID to measure the level of SpoIIID during sporulation of wild-type cells, mutants defective in sigma K production, and a mutant engineered to produce sigma K earlier than normal. The feedback loop operates at the level of synthesis and/or stability of spoIIID mRNA, as demonstrated by measuring the level of spoIIID mRNA during sporulation of wild-type cells and mutants defective in sigma K production. Our results suggest that a rise in the level of sigma K during the stage (IV) of spore cortex formation causes a decrease in the level of SpoIIID, which, at least in part, establishes the switch to the stage V (spore coat formation) pattern of mother-cell gene expression.
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PMID:Fate of the SpoIIID switch protein during Bacillus subtilis sporulation depends on the mother-cell sigma factor, sigma K. 146 17

The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway. gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set. The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively). We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC. We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K. These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes. In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K. The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.
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PMID:Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes. 151 43


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