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 human class II transcription factor TFIIB (rTFIIB) was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified rTFIIB is identical to the endogenous factor according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to recognize the stable preinitiation complex formed between TFIID and the adenovirus 2 major late TATA box as demonstrated by gel shift as well as by DNase I footprinting assays, and transcription activity.
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PMID:Expression in Escherichia coli: purification and properties of the recombinant human general transcription factor rTFIIB. 145 51

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

By using a DNase I footprinting assay, we have purified a factor by DNA affinity chromatography that binds to the minimal enhancer region of the Drosophila knirps gene and subsequently identified the protein as the core histone H2B. This inadvertent purification of a core histone as a putative sequence-specific DNA binding protein was due to a previously unknown property of H2B to interact with DNA in a periodic manner. Moreover, we found that each of the individual core histones, but not histone H1 or high mobility group protein 1, bound to the knirps enhancer to give a repetitive DNase I footprint pattern with a periodicity of about 10 base pairs, which is approximately one turn of the DNA helix. In addition, preparations containing the core histones H2A-H2B or H3-H4 yielded identical periodic DNase I footprint patterns on several different promoter and enhancer regions. These findings suggest that there are periodic, homotypic interactions between DNA-bound core histones that result from an alteration of the overall DNA structure such as the curvature rather than a specific sequence. We have also shown that histones H2A-H2B can repress initiation of transcription by RNA polymerase II. The phenomena described here may reflect histone-DNA interactions in non-nucleosomal stretches of chromatin and could be involved in some aspects of either rotational or translational positioning of nucleosomes. Furthermore, these findings indicate that a repeated 10 bp DNase I ladder, which has previously been considered to be a property of an intact nucleosome, can also be generated with subnucleosomal components. It will thus be necessary to reevaluate the criteria applied to the analysis of nucleosomes both in vivo and in vitro.
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PMID:Periodic binding of individual core histones to DNA: inadvertent purification of the core histone H2B as a putative enhancer-binding factor. 148 Apr 89

The general transcription factor TFIIIC is necessary for transcription initiation by RNA polymerase III. TFIIIC binds predominantly to the B-Block promoter element, which is present in tRNA genes, several viral RNA genes and repetitive DNA elements, and to the TFIIIA.DNA complex on 5 S RNA genes. Here we report a characterization of Xenopus laevis TFIIIC and its interaction with the TFIIIA.5 S RNA gene complex. A polypeptide with apparent molecular mass of 85 kDa was specifically cross-linked to a B-Block oligonucleotide by UV light. This polypeptide was present in the partially purified TFIIIC fraction and in a complex with a B-Block double-stranded oligonucleotide isolated by nondenaturing gel electrophoresis. TFIIIC.TFIIIA.DNA gel mobility shift complexes were obtained using B-Block DNA affinity-purified TFIIIC and buffer conditions employing low Mg2+ (1 mM) and high dithiothreitol (7 mM) concentrations. Three TFIIIC.TFIIIA.5 S RNA gene complexes were observed by gel mobility shift analysis. One of these complexes was resistant to dissociation by the addition of competing DNA, but the formation of all three complexes was prevented by the inclusion of excess specific competitor DNA in the initial binding reactions. The apparent affinity of TFIIIC for the TFIIIA.5 S DNA complex was 5-fold higher for the somatic-type 5 S RNA gene than for the oocyte-type 5 S RNA gene. Mutations near the 5' boundary of the TFIIIA binding site alter the DNase I footprint of the TFIIIA.DNA complex and reduce the affinity of TFIIIA-mutant 5 S gene complexes for TFIIIC. Differences in TFIIIC affinity for the two classes of 5 S RNA genes may play a role in the developmental regulation of these gene families.
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PMID:Interaction of Xenopus TFIIIC with the TFIIIA.5 S RNA gene complex. 151 47

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.
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PMID:Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter. 153 52

Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to -54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C. We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.
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PMID:Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region. 153 36

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A. Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo. We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein.
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PMID:Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE. 155 84

The structure and properties of ternary complexes of RNA polymerase are of central importance in understanding the mechanisms of transcriptional elongation and termination, and the regulation of these primary steps in gene expression. However, there has been no systematic study of the structure and properties of such complexes along a single transcription unit. Recently, we have described the isolation of a collection of halted ternary complexes of Escherichia coli RNA polymerase bearing transcripts from 11 to 35 nucleotides in length along two different transcription units (accompanying paper). Here, we report structural studies of these complexes using DNase I footprinting. Surprisingly, nearly all of the different ternary complexes have distinctly different footprints along the two DNA strands, and the position of the footprint relative to the 3' end of the transcript also varies for most complexes. Halted complexes bearing transcripts of comparable size do not have identical footprints; hence, DNA sequence as well as transcript length plays a role in determining the size and position of the footprint. These differences in structure are consistent with our earlier findings that ternary complexes can differ considerably in stability and gel mobility. The downstream boundary of the RNA polymerase in ternary complexes does not move forward regularly as successive nucleotide residues are added to the RNA chain. In contrast, the upstream boundary moves forward more or less in concert with the movement of the 3' terminus of the transcript. These factors lead to a general compression of the overall footprint as transcription proceeds, together with a steady movement of the 3' terminus of the RNA toward the downstream boundary of the polymerase. Ultimately, after the length of the RNA transcript has increased from eight to ten nucleotides, the downstream boundary of the complex is found to move downstream along the DNA, suggesting a translocation event. We suggest that RNA chain elongation, like RNA chain initiation, may involve a saltatory process in which net translocation of the complex along the DNA occurs only after addition of a number of ribonucleotides to the RNA chain.
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PMID:Structural analysis of ternary complexes of Escherichia coli RNA polymerase. Deoxyribonuclease I footprinting of defined complexes. 159 19

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.
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PMID:Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii. 162 Jun 19

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.
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PMID:Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. 165 94


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