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 alternative sigma factor sigma 54 of enteric bacteria, or its homologue in other purple bacteria, is required for transcription of genes whose products have diverse physiological roles. Previous studies have indicated that sigma 54 confers on core RNA polymerase the ability to recognize a specific class of promoters but not the ability to isomerize from closed to open complexes. Isomerization requires ATP and one member of a family of activator proteins, it being different activator proteins that allow this form of polymerase to respond to different physiological signals. We have developed a strategy for overproducing and purifying sigma 54 from Salmonella typhimurium and have studied several biochemical properties of reconstituted sigma 54-holoenzyme. The initial binding constant KB for the formation of closed complexes between this holoenzyme and the ginA promoter in our transcription buffer is approximately 3 x 10(8) M-1, which was determined from DNaseI protection assays at 37 degrees C. After the formation of open complexes, several properties of sigma 54-holoenzyme appear to be similar to those of sigma 70-holoenzyme. We have determined the complete nucleotide sequence of the gene encoding sigma 54 (ntrA) in Salmonella.
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PMID:Purification of the alternative sigma factor, sigma 54, from Salmonella typhimurium and characterization of sigma 54-holoenzyme. 191 63

The inhibitory effect of esters of p-hydroxybenzoic acid (kelletinins I and A), extracted from the marine gastropod Buccinulum corneum, have been tested on eukaryotic and prokaryotic enzymes of DNA metabolism such as DNA polymerases alpha and beta, DNA polymerase I, Exo III, pancreatic DNAse I, micrococcal DNAse and E. coli RNA polymerase. Kelletinin I and kelletinin A inhibit preferentially DNA polymerase alpha. The inhibitory effect of kelletinin I involves the hydroxyl group of p-hydroxybenzoic acid.
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PMID:Kelletinin I and kelletinin A from the marine mollusc Buccinulum corneum are inhibitors of eukaryotic DNA polymerase alpha. 199 46

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.
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PMID:RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. 248 70

The transcription initiation site of the mel operon from Streptomyces glaucescens, determined by S1 mapping and primer elongation experiments, lies 32 to 34 bp upstream of the translation initiation codon of the first open reading frame. A total of 172 to 219 bp upstream of the transcription start point are necessary for a fully active and regulated mel promoter. Deletion analysis, gel retardation assays and DNAse I footprint experiments facilitated division of the promoter into three functional domains, which include the RNA polymerase recognition site up to nucleotides -33 to -42, the binding region of a protein of assumed regulatory function between nucleotides -65 and -93, and an upstream activator site, located between positions -158 and -219.
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PMID:Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens mel operon. 260 68

The sequence specificity of daunomycin was assessed using competition equilibrium dialysis, DNAse I footprinting and an E. coli RNA polymerase transcription inhibition assay; similar studies were performed on adriamycin and a new bis-intercalating daunomycin dimer. The results clearly demonstrate that the highest affinity sites are CA for daunomycin and adriamycin, and CACA for the bis-daunomycin. Other modest affinity (GC, CG, CT, TC, CC, AC) and poor affinity binding sites (AA, AT, TA) were also observed. Our results are in agreement with (a) the observed 5'-pyrimidine-purine-3' sequence preference of intercalating drugs, (b) the reported role played by OH(9) of daunomycin in the stabilization of the drug/DNA intercalation complex, and (c) the thermodynamics of nearest neighbour base-pair unstacking at the intercalation site. The CA specificity of daunomycin and adriamycin suggests that their biological activity may arise from association with the CA containing sequences which are thought to be associated with genetic regulatory elements in eukaryotes. The implications for future anthracycline drug design are presented in this context.
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PMID:Elucidation of the DNA sequence preferences of daunomycin. 285 76

Yeast mitochondrial RNA polymerase can bind specifically to promoter-containing DNA fragments in vitro as detected by DNAse I or methidiumpropyl-EDTA. Fe(II) protection assays and gel retardation experiments. Retardation of RNA polymerase-DNA complexes was most pronounced when the promoter was located in the middle of a DNA fragment and was diminished when RNA polymerase was bound near one of the ends. This indicates that upon RNA polymerase-binding the DNA undergoes a conformational change which is most likely a bend. The degree of introduced bending correlated with the efficiency of transcription and promoter-binding in a series of promoter mutants, suggesting that bending is a functional event during promoter utilisation.
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PMID:RNA polymerase induces DNA bending at yeast mitochondrial promoters. 305 Aug 96

This paper reports three experiments concerning the structural relationship between the Xenopus transcription factor IIIA (TFIIIA), the histone octamer and the Xenopus somatic gene for 5S RNA. Quantitative footprinting methods have been used in order to discover where and how TFIIIA and the histone octamer bind to the same gene independently and also in a triple complex. First, DNaseI and DNaseII protection experiments show that TFIIIA binds to positions 45-97 within the gene, in agreement with other workers. Second, the histone octamer takes up a unique, well-defined position with respect to DNA sequence. The nucleosome core extends to position 78 of the gene and therefore overlaps the TFIIIA binding region by approximately 35 bp. Third, it is shown that a triple complex can be formed between TFIIIA, the histone octamer and the 5S RNA gene. TFIIIA displaces the DNA from the histone surface in the 35-bp region of overlap. This has led to a three-dimensional model which explains how RNA polymerase III could interact simultaneously with transcription factors bound at the internal control region of the 5S RNA gene and the start point of transcription. The model also explains how histone H1 could repress transcription of 5S RNA genes.
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PMID:Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. 409 86

Electron microscope spread preparations of nuclear chromatin derived from early S-phase of Physarum reveal 'beads on a string' for nonreplicated and a portion of newly replicated chromatin. Many of the early replicons contain transcription units as visualized by nascent transcripts. They are, in most cases, arranged in continuous length gradients on both newly replicated strands of chromatin, the putative origin of replication being within the transcription unit. Preferential release of DNA as acid precipitable material by DNAse I and of RNA polymerase B (estimated as release of labeled alpha-amanitin bound to isolated nuclei) is observed in early S-phase, but only if DNA synthesis is not inhibited. Also, generation of a small particle (peak A) by staphylococcal nuclease, characteristic of transcriptionally active chromatin, depends on concomitant replication of early replicons. It is concluded that DNA replication is a prerequisite for its transcription by RNA polymerase B. Thus, the sequential replication of the genome of Physarum dictates the order of transcription during S-phase which may in part control the proliferative mitotic cycle of Physarum.
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PMID:Physical relationship between replicons and transcription units in Physarum polycephalum. 621 73

Nuclei were prepared from mature and immature hen erythrocytes and incubated for RNA synthesis in the absence or in the presence of Sarkosyl. The in vitro labelled synthesized RNA was hybridized to specific 5' and 3' fragments of the chicken adult beta-globin gene to investigate the possible presence of RNA polymerase molecules bound to this gene in the form of transcriptional complexes. Surprisingly, such RNA polymerase B molecules were found located preferentially in the 5' end moiety of the beta-globin genes of mature erythrocytes, although they are apparently evenly distributed along the beta-globin genes of immature polychromatic erythrocytes. The significance of these observations with respect to (1) preferential DNaseI sensitivity of "genes which have been transcribed" and (2) control of transcription in eukaryotic cells is discussed.
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PMID:Clustering of RNA polymerase B molecules in the 5' moiety of the adult beta-globin gene of hen erythrocytes. 626 56

Activation of the spoIIG promoter at the onset of sporulation in Bacillus subtilis requires the regulatory protein, Spo0A, which binds to two sites in the promoter, sites 1 and 2. Phosphorylation of Spo0A is essential for the initiation of sporulation. Therefore, we examined the role of Spo0A phosphorylation in spoIIG promoter activation. Phosphorylation of Spo0A stimulated transcription from the spoIIG promoter in vitro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of Spo0A increased its affinity for site 2 more than for site 1, which is the site to which nonphosphorylated Spo0A binds most avidly. This result could not be explained by increased cooperativity between Spo0A bound at sites 1 and 2 because the increased affinity for site 2 by phosphorylated Spo0A was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located Spo0A-binding sequences in the spoIIG promoter by DMS protection assays and mutational analysis, and found that site 1 contains one higher-affinity binding sequence whereas site 2 contains two weaker-binding sites. Two substitutions in site 2 of the spoIIG promoter that change the sequence to be more like an optimal Spo0A-binding site were found to increase promoter activity. Moreover, phosphorylation of Spo0A was not required in vivo for activation of the spoIIG promoter containing these strong binding sites. The results suggest that the primary role for phosphorylation of Spo0A is to increase its affinity for specific sites rather than to activate an activity of Spo0A that acts on RNA polymerase at promoters.
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PMID:Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. 828 22


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