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 promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli gene aroG (encoding the phenylalanine-sensitive 3-deoxy-arabinoheptulosonate-7-phosphate synthase) were located. Primer extension analysis and nuclease S1 mapping of in vivo transcripts were used to determine the 5' and 3' ends, respectively, of the mRNA. Both ends exhibited some heterogeneity with respect to length. The 3' end of the major molecular species was located within a region that has structural homology with known rho-independent terminators. The location of the aroG promoter was identified in both strands of the DNA by in vitro DNase I footprinting and methylation protection experiments with RNA polymerase. In these experiments, a region of up to 80 base pairs (bp) was protected by the binding of RNA polymerase. The location of the aroG operator was also identified in both strands of the DNA by in vitro DNase I footprinting with pure TyrR. TyrR protected 26 to 28 bp of DNA containing a 22-bp palindrome (TYR R box) and overlapping the -35 region of the promoter. Mutations in the aroG regulatory DNA were isolated by site-directed mutagenesis and cloned in a low-copy-number plasmid to generate aroG-lac fusions. The effects of the mutations on the regulation of aroG expression were determined by measuring the beta-galactosidase activities of the fusions in strains with tyrR, tyrR+, and multicopy tyrR+ genotypes. The results of this mutant analysis confirmed that the aroG operator contains a single TYR R box.
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PMID:Identification of the promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG. 197 May 63

The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr). We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199. Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.
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PMID:Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites. 198 82

The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central role in the uptake of exogenous long-chain fatty acids. The nucleotide sequence of the fadL gene revealed a single open reading frame of 1,344 bp encoding a protein with 448 amino acid residues and a molecular weight of 48,831. The transcriptional start, analyzed by primer extension, was shown to be 95 bp upstream from the translational start. Apparent -10 and -35 regions were found at -12 and -37 bp upstream from the transcriptional start. Three regions with hyphenated dyad symmetry (two between the transcriptional start and the translational start and one upstream from the -10 and -35 regions) were identified that may play a role in the expression of fadL. The protein product of the fadL gene contained a signal sequence and signal peptidase I cleavage site similar to that defined for other E. coli outer membrane proteins. The N-terminal sequence of mature FadL protein was determined by automated amino acid sequencing of protein purified from the outer membrane of a strain harboring fadL under the control of a T7 RNA polymerase-responsive promoter. This amino acid sequence, Ala-Gly-Phe-Gln-Leu-Asn-Glu-Phe-Ser-Ser, verified the signal peptidase I cleavage site on pre-FadL and confirmed the N-terminal amino acid sequence of FadL predicted from the DNA sequence. Mature FadL contained 421 amino acid residues, giving a molecular weight of 45,969. The amino acid composition of FadL deduced from the DNA sequence suggested that this protein contained an abundance of hydrophobic amino acid residues and lacked cysteinyl residues. The hydrophobic amino acids within FadL were predicted to contribute to at least five regions of the protein with an overall hydrophobic character. The amino acid sequence of FadL was used to search GenBank for other proteins with amino acid sequence homology. These data demonstrated that FadL and the heat-modifiable outer membrane protein P1 of Haemophilus influenzae type b were 60.5% conserved and 42.0% identical over 438 amino acid residues.
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PMID:Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. 198 39

The specificity of regulation by attenuation of the ilvGMEDA operon of Escherichia coli was examined by making alterations in the peptide-coding portion of the leader region. The effects of the alterations on attenuation control were monitored by operon fusions with the lacZ or cat gene. Substitution of the tandem leucine codons with arginine codons did not result in arginine control of attenuation even though the altered leader transcripts contained three consecutive arginine codons. Substitution of the single leucine codon with a proline codon at position 10 of the putative peptide, which had been shown to be important in the regulation of the Serratia marcescens ilv operon, did not result in control of attenuation by proline. Since the formation of neither proline nor arginine biosynthetic enzymes is regulated by attenuation control, the effect of tandem phenylalanine codons in place of the tandem leucine codons was examined and found not to result in control by phenylalanine supply. The latter failure may have been due to a configuration in the secondary structure of the protector stem of the leader transcript different from that of the wild-type transcript. The results of the study favored the idea that the lead ribosome does not initiate translation of the leader transcript until after the RNA polymerase has reached the pause site (117 bases into the leader region).
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PMID:Mutations replacing the leucine codons or altering the length of the amino acid-coding portion of the ilvGMEDA leader region of Escherichia coli. 200 56

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.
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PMID:Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten. 213 24

An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.
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PMID:In vitro suppression of an amber mutation by a chemically aminoacylated transfer RNA prepared by runoff transcription. 230 38

The effect of infusion of a methionine-free total parenteral nutrition solution for 7 d on ribonucleic acids in liver of rats were investigated. The control solution contained leucine, valine, isoleucine, lysine, phenylalanine, threonine, tryptophan, arginine, histidine, glycine, methionine, glucose and vitamins and minerals. Deprivation of a methionine is known to increase the activity of RNA polymerase I. Infusing the methionine-free solution resulted in the accumulation of RNA molecules larger than 28S in the liver nuclei and resulted in a higher rate of rRNA synthesis than in rats infused with the control solution. A methionine deficiency did not impede either the processing of 45S pre-rRNA or transport of 28S and 18S rRNA into cytoplasm. When rats were infused with the methionine-free solution for 7 d followed by the control solution for 2 d, the level of RNA in the nucleus as well as the rate of RNA polymerase I were similar to the levels in rats receiving the control solution for 9 d. There were no significant changes in the rate of DNA synthesis due to nutritional manipulations.
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PMID:Alteration in the ribonucleic acids in rat liver induced by a methionine-free total parenteral nutrition solution. 243 90

Full-length cDNA copies of mRNAs coding for the matrix (M) proteins of vesicular stomatitis virus and its mutant tsO23(III) were cloned in pBSM13- (BlueScribe). The authenticity of these clones was demonstrated by restriction enzyme mapping, DNA sequencing, and in vitro transcription and translation to identify the two M proteins by Western immunoblotting with epitope-specific monoclonal antibodies. Site-directed mutants were constructed by primer extension of synthetic oligodeoxynucleotides with one or two nucleotide changes to alter the glycine at amino acid 21 of the wild-type (wt) M gene to glutamic acid, alanine, or proline. Similarly, a revertant was created in the M gene of mutant tsO23 by a Glu-21----Gly substitution. A series of wt- and mutant-M-gene chimeras was also constructed to create mutant and revertant clones with Leu----Phe and His----Tyr alterations at amino acids 111 and 227, respectively. We then moved the wt and tsO23 M genes and their site-specific mutants and chimeras cloned in pBSM13- into the eucaryotic expression vector pTF7 directed by the T7 bacteriophage RNA polymerase of the vaccinia virus recombinant vTF1-6,2. Western blot analysis of the M proteins transiently expressed in CV-1 cells by plasmids carrying M genes altered at amino acid 21 revealed that the critical antigenic determinant (epitope 1) is expressed only by the Gly-21 M protein and not by Glu-21, Ala-21, or Pro-21 M proteins. Of particular interest is an apparent conformational change, evidenced by slightly but significantly retarded electrophoretic migration, in plasmid-expressed M proteins with amino acids substituted for glycine at position 21. The glutamic acid at position 21 of tsO23 is not responsible for its temperature-sensitive phenotype, because a tsO23 revertant plasmid with glycine substituted at position 21 fails to rescue tsO23 virus in cells infected at the restrictive temperature; conversely, plasmids expressing wt M protein with substitutions of glutamic acid, alanine, or proline at position 21 are just as effective in marker rescue of tsO23 as is the Gly-21 wt M protein. Marker rescue experiments with wt- and mutant-M-gene chimeras support the hypothesis of K. Morita, R. Vanderoef, and J. Lenard (J. Virol. 61:256-263, 1987) that the temperature-sensitive phenotype of tsO23 is due to a phenylalanine substituted for leucine at amino acid 111, rather than the His-227----Tyr substitution or the Gly-21----Glu substitution, which independently accounts for the loss of epitope 1 in the mutant M protein of tsO23.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Site-specific mutations in vectors that express antigenic and temperature-sensitive phenotypes of the M gene of vesicular stomatitis virus. 245 88

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.
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PMID:Mutant rho factors with increased transcription termination activities. II. Identification and functional dissection of amino acid changes. 247 57


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