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)

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

Incubation of the E. coli RNA polymerase with a polypeptide factor from the protozoan Tetrahymena reduces the affinity of the holoenzyme for DNA. SDS-polyacrylamide gel electrophoresis of the peptide-treated RNA polymerase showed that the band pattern of the polymerase subunits was strongly altered. The three large subunits, beta', beta and sigma, disappear and a high number of rapidly migrating bands appeared. However, a brief heat treatment of the samples almost restored the original RNA polymerase subunit composition, and in addition a high molecular weight protein band approximately 240 kDa appeared. It is suggested that the Tetrahymena peptide specifically binds to the RNA polymerase and changes the structures of the large subunits.
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PMID:A peptide from Tetrahymena disrupts subunit organization of E. coli RNA polymerase. 147 89

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis lambda gt11 cDNA library with mono-specific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98-100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The cDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human cleavage signal-1 protein; cDNA cloning, transcription and immunological analysis. 155 70

The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
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PMID:Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro. 176 63

We have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter phi 10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.
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PMID:Expression of active yeast pyruvate decarboxylase in Escherichia coli. 179 34

Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET-8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross-linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C-terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83-152, 117-186 and 185-240) had little effect on the oligomerization. Co-expression of the N- and C-terminal mutant clones in vitro did not produce oligomers. These results indicate that intra-molecular interactions with both the N- and C-terminal domains are essential for oligomer formation.
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PMID:Formation of nucleophosmin/B23 oligomers requires both the amino- and the carboxyl-terminal domains of the protein. 191 43

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
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PMID:E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription. 192 88

The complete sequence of the type VI dihydrofolate reductase (DHFR) gene from plasmid pUK672 was determined. The structural gene coded for a polypeptide of 157 amino acids which had a deduced mol. wt of 17,424. Comparison with amino-acid sequences of the type I, type V and Escherichia coli K12 chromosomal DHFRs showed that there was 63%, 61% and 31% homology respectively. Putative RNA polymerase and ribosomal binding sites were identified proximal to the initiation codon and a feature consistent with transcription termination was present distal to the coding region. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the enzyme had a subunit mol. wt of 17,500.
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PMID:Nucleotide sequence of dihydrofolate reductase type VI. 194 91

RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.
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PMID:Sequencing and expression of the rne gene of Escherichia coli. 201 93

Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase. RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.
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PMID:Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. 201 26


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