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)

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.
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PMID:In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator. 190 59

E. coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partial cyanogen bromide cleavage of the labelled polypeptide afforded a series of "single-hit" labelled peptides, the electrophoretic pattern of which suggested that the labelling site was His1237. This conclusion was confirmed by a similar pattern obtained with products of the cyanogen bromide cleavage of a radioactive peptide obtained by the limited trypsinolysis (C-terminal peptide consisting of 423 amino acid residues). Interpretation of our earlier results in favour of His1116 as the labelling point (Dokl. Acad. nauk SSSR, 1985, v. 281, p. 723) was incorrect due to the electrophoretic "compression" of three labelled peptide bands.
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PMID:[Localization of a histidine residue in the binding site for the initiating substrate of E. coli RNA-polymerase]. 331 73

Mammalian cells contain two subspecies of RNA polymerase II, designated IIO and IIA. The objectives of these studies were to determine the structural relationship between these subspecies and to determine the functional significance of these differences. Subunits IIo and IIa were purified from calf thymus, and the effect of alkaline phosphatase treatment on electrophoretic mobility and immunochemical reactivity was examined. The removal of phosphate converts subunit IIo to a form indistinguishable from that of subunit IIa. These results indicate that subunit IIo is produced by multisite phosphorylation of subunit IIa. The distribution of phosphate within subunit IIo was determined by CNBr cleavage of in vivo labeled HeLa cell RNA polymerase II. 32P-Labeled subunit IIo was purified by immunoprecipitation and cleaved with CNBr, and the resultant peptides were analyzed. The quantitative recovery of 32P in the C-terminal peptide establishes that this domain is the primary site of phosphorylation. In an effort to assess the level of phosphorylation of the transcriptionally active form of RNA polymerase II in HeLa nuclei, transcription was carried out in the presence of 4-thiouracil triphosphate and the nascent labeled transcript cross-linked to RNA polymerase. Specific photoaffinity labeling of subunit IIo was observed. Alkaline phosphatase treatment results in an increase in the mobility of photoaffinity labeled subunit IIo to approach that of subunit IIa. These results indicate that subunit IIo is a component of transcriptionally active RNA polymerase II.
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PMID:Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II. 362 68

The anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.
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PMID:The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein. 841 14

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.
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PMID:Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis. 862 54

Colicin V (ColV), an antibacterial peptide toxin, uses a dedicated signal sequence-independent export system for its extracellular secretion in Escherichia coli. The products of at least three genes (a chromosomal tolC gene and two plasmid-born cvaA and cvaB genes) are involved in this process. To characterize the gene products, the cvaA gene was subcloned and expressed under the control of T7 RNA polymerase promoter. Two in-frame proteins, CvaA and CvaA*, were expressed and identified. DNA sequences predicted that both proteins have two potential translational initiation sites. N-terminal peptide sequencing showed that the translation of CvaA starts from a TTG, 11 amino acids upstream of the previously proposed ATG initiation site. CvaA* is translated from an upstream ATG. Expression of both CvaA and CvaA* was induced by the iron chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulated at least partially by Fur. CvaA*-depleted cells were found to secrete less ColV, based on reduced activity in the supernatant, than did wild type, which was recovered by the addition of a plasmid producing CvaA*. Interestingly, CvaA*-depleted and wild-type cells had similar levels of intracellular ColV activity. Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA* depletion. However, CvaA in CvaA*-depleted cells was less stable than that in wild-type cells, indicating that CvaA* may directly or indirectly affect the stability of CvaA. We conclude that CvaA* is not essential for ColV secretion but that it enhances the ColV secretion by stabilizing the CvaA protein.
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PMID:Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion. 900 22

We have undertaken a characterization of the CM2 protein of influenza C virus. The CM2 coding region of RNA segment 6 (nucleotides 731-1147) was cloned from two strains of influenza C virus and expressed using the vaccinia virus-bacteriophage T7 RNA polymerase (vac-T7) system. An antiserum raised to a C-terminal peptide in the CM2 open reading frame recognized the CM2 protein in influenza C virus-infected cells and after vac-T7 expression of the CM2 open reading frame. CM2 is posttranslationally modified by addition of high-mannose carbohydrate chains (Mr approximately 18 kDa) and by further addition of polylactosaminoglycans (Mr approximately 21-35 kDa). The available data indicate that CM2 has a cleavable signal peptide at the N-terminus of the protein. Site-directed mutagenesis eliminated the single potential N-linked carbohydrate attachment site on CM2 and indicated that the protein has an NoutCin orientation in membranes. Nonreducing SDS-PAGE indicated that the protein was expressed as disulfide-linked dimers and tetramers. Cell surface biotinylation and indirect immunofluorescence showed the protein to be expressed at the cell surface. Elimination of the N-linked carbohydrate attachment site and addition of a C-terminal HA epitope tag did not adversely affect surface expression of CM2. The NoutCin membrane orientation of CM2, the size of the ectodomain and cytoplasmic tail of CM2, and its ability to form disulfide-linked oligomers are reminiscent of the structural properties of influenza A virus M2 and influenza B virus NB proteins.
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PMID:The CM2 protein of influenza C virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. 935 55

Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183. Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition.
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PMID:Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor. 1201 19

In the protist parasite Trypanosoma brucei, RNA polymerase (pol) I transcribes the large ribosomal RNA gene unit and, in addition, variant surface glycoprotein gene expression sites and procyclin gene transcription units. The multifunctional role of RNA pol I in this organism is unique among eukaryotes, but only its largest subunit TbRPA1 has been characterized thus far. We have recently established the procyclic cell line RPIC which exclusively expresses RNA pol I tagged with the protein C epitope at the TbRPA1 C-terminus. In the present study, we prepared RPIC cell extracts and immunopurified RNA pol I using anti-protein C affinity matrix under high stringency conditions. We were able to identify five specific polypeptides on a silver-stained polyacrylamide-SDS gel with apparent molecular weights of 200, 180, 55, 29, and 22 kDa. Interestingly, the second largest subunit, TbRPA2, is 42-58 kDa larger than counterparts of other organisms. We have cloned and sequenced the complete TbRPA2 cDNA and found an open reading frame for a polypeptide of 179.5 kDa. The deduced amino acid sequence of TbRPA2 contains a unique N-terminal domain of approximately 250 amino acids. By raising a polyclonal antibody against a N-terminal peptide sequence of TbRPA2, we could specifically detect this polypeptide in immunoblots showing that it co-purifies with epitope-tagged TbRPA1. Moreover, we identified the homologous gene sequence LmRPA2 in Leishmania major and found that it encodes a homologous extension domain. Therefore, the N-terminal extra domain in trypanosomatid RPA2 polypeptides may serve a parasite-specific function.
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PMID:The second largest subunit of Trypanosoma brucei's multifunctional RNA polymerase I has a unique N-terminal extension domain. 1261 18

Beacon is a 73-amino acid peptide encoded by a novel gene in the hypothalamus of Israeli sand rat Psammomys obesus. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical techniques were used to investigate the presence of beacon mRNA and the distribution of beacon-immunoreactivity (irBC) in the hypothalamus of ICR mice. RT-PCR experiments revealed beacon mRNA in the mouse hypothalamus. Using a rabbit polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73), irBC was detected in the mouse hypothalamus and pituitary. In the hypothalamus, irBC was concentrated in perikarya of the supraoptic (SO), paraventricular (PVH) and accessory neurosecretory nuclei and in cell processes of the median eminence and pituitary stalk. In the pituitary, irBC was noted mainly in the posterior lobe. Double-labeling the hypothalamic sections with guinea-pig vasopressin-antiserum or mouse monoclonal oxytocin-antibody and beacon-antiserum revealed that <30% of vasopressin-immunoreactive neurons and nearly all oxytocin-immunoreactive neurons in the PVH and SO were irBC. The result shows the presence of beacon mRNA in the mouse hypothalamus, and the distribution of irBC is distinctively different from that reported in the hypothalamus of Psammomys obesus, but similar to that of the Sprague-Dawley rats described in our earlier study. More interestingly, Blast search uncovered a 73-amino acid peptide, human ubiquitin-like 5, which has the same exact sequence as beacon. Thus, irBC observed in the mouse brain could be that of ubiquitin-like 5.
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PMID:Beacon/ubiquitin-like 5-immunoreactivity in the hypothalamus and pituitary of the mouse. 1293 56


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