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)

A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue.
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PMID:Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system. 212 Jan 83

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).
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PMID:Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii. 212 Jan 86

Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late. The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription. A plasmid containing a Pm-lacZ operon fusion was constructed. beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction. Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17. Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator. Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insertion of a linker into mor substantially reduced the ability of Mu to form plaques. When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu. The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression. Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology.
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PMID:Identification of a positive regulator of the Mu middle operon. 214 78

Specific transcription complexes were formed with yeast RNA polymerase I using a cognate oligoribotri-nucleotide primer (GCG) to initiate transcription on short synthetic single-stranded DNA templates. The templates were designed to limit the incorporation of a photoprobe, 4-thiouridine triphosphate, to a single unique position at the 3' terminus of the product RNA (position 12, 13, 14, or 15). The resulting transcription complexes were photolyzed to cross-link the bound transcript (radiolabeled with [alpha-32P]CTP) to the protein with the probe located at the catalytic site. Separation of the protein subunit components by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by autoradiography and silver staining revealed that the two largest subunits (A190 and A135) were radiolabeled. The ratio of subunit labeling (A190/A135) decreased as the RNA transcript increased from 12 to 15 nucleotides in length. This decrease in ratio resulted from a progressive reduction of A190 subunit labeling while the A135 subunit derivatization remained essentially constant. It was also observed that the DNA template was radiolabeled.
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PMID:Yeast RNA polymerase I. Derivatization of the 190 and 135 subunits by 4-thiouridine monophosphate positioned uniquely at the 3' terminus of an enzyme-bound 32P-containing transcript initiated by a triribonucleotide primer on synthetic single-stranded DNA. 215 57

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].
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PMID:Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system. 215 86

The human gastric (H+ + K+)-ATPase gene (15 kilobases) was cloned, and its nucleotide sequence was determined. The gene has 22 exons and codes a protein of 1,035 residues including the initiator methionine (Mr = 114,047). A conserved lysine-rich sequence with inserted glycine residues was found near the amino terminus of the enzyme. The phosphorylation site and pyridoxal 5'-phosphate- and fluorescein isothiocyanate-binding residues found in the rat and pig enzymes are also conserved in the human enzyme. The positions of introns in the human (H+ + K+)-ATPase gene are essentially the same as those in the human (Na+ + K+)-ATPase alpha and alpha III subunits; but the first introns of the two enzymes are difficult to align, and unlike in the (Na+ + K+)-ATPase gene, the sixth exon in the (H+ + K+)-ATPase gene is not separated by an intron. Furthermore, the ninth intron is located two bases upstream of the position for the corresponding intron of the (Na+ + K+)-ATPase alpha III subunit. The similarity in organization of these two ATPase genes and the homology in the primary structures of their proteins (approximately 60%) suggest that these two genes were derived from a common ancestral gene. However, the 5'-flanking regions of the genes for (H+ + K+)-ATPase and the (Na+ + K+)-ATPase alpha (+) subunit show no apparent sequence homology, indicating that their transcriptions are regulated differently. The control region of the fast-twitch sarcoplasmic reticulum Ca2(+)-ATPase gene also showed no sequence homology to that of (H+ + K+)-ATPase. The 5'-flanking region of the (H+ + K+)-ATPase gene contains potential binding sites for RNA polymerase II and various transcriptional regulation factors and several direct and inverted repeat sequences which may be important for specific and controlled expression of the gene in gastric parietal cells. There are two polyadenylation signals in the 3'-flanking region of the (H+ + K+)-ATPase gene, but the sequence of this region shows no homology to those of the corresponding regions of the genes for the (Na+ + K+)-ATPase alpha and alpha III subunits.
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PMID:Human gastric (H+ + K+)-ATPase gene. Similarity to (Na+ + K+)-ATPase genes in exon/intron organization but difference in control region. 216 Sep 52

By use of techniques described recently for lac permease [Roepe, P.D., & Kaback, H.R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6087], the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system. As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion. Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea. Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea. Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport. The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins.
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PMID:Isolation and functional reconstitution of soluble melibiose permease from Escherichia coli. 218 31

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate-polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62,000; nagA, 45,000; nagB, 29,000. In addition, another product of Mr 44,000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weight (MWs) of the gene products derived from the sequence are as follows: nagA, 40,954; nagB, 29,657; nagC, 44,664; nagE, 68,356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27,181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: nagD nagC nagA nagB nagE.
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PMID:Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli. 219 Jun 15

Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B). 219 20

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
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PMID:Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand. 228 Jul 70


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