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)

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.
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PMID:Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. 190 71

Variants with mutations in three parts of the tRNA-like structure of turnip yellow mosaic virus RNA (the anticodon, the discriminator position in the amino acid acceptor stem, and in the variable loop) were created via site-directed mutagenesis of a cDNA clone and transcription with T7 RNA polymerase. The valylation properties of transcripts were studied in the presence of pure yeast valyl-tRNA synthetase. Mutation of the central position of the anticodon triplet resulted in a quasi-total loss of valylation activity, indicating that the anticodon is a principal determinant for valylation of the turnip yellow mosaic virus tRNA-like structure. These anticodon mutants interacted with yeast valyl-tRNA synthetase with affinities comparable to those of the wild-type RNA and behaved as competitive inhibitors in the valylation reaction of yeast tRNAVal. The defective aminoacylation of these mutants therefore results from kinetic rather than affinity effects. Minor negative effects on valylation efficiency were observed for mutants with substitutions at the two other sites studied, suggesting a structural role or a limited contribution to the valine identity of the tRNA-like molecule.
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PMID:Specific valylation identity of turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase is directed by the anticodon in a kinetic rather than affinity-based discrimination. 199 71

Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented. Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling. Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation). The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities. Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed. While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.
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PMID:Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs. 220 41

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

Previous studies on two Escherichia coli rpoB mutants, carrying single amino acid substitutions at approximate amino acid positions 736 and 906 in the beta subunit, showed that these alterations in the RNA polymerase resulted in an apparent reduced response to valine-induced amino acid starvation in vivo and prevented ppGpp-mediated inhibition of transcriptional initiation at stable RNA promoters in vitro. These observations suggested that the mutations had altered either the ppGpp binding site or the promoter selectivity of the enzyme. The in vivo analysis presented here indicates that these mutants encode an RNA polymerase that responds normally to changes in the level of ppGpp; their apparent relaxedness is due to a reduced accumulation of ppGpp during isoleucine starvation. Thus, there is no indication that the mutations have altered ppGpp binding sites. These observations and the difference between in vitro and in vivo results can be explained by the assumption that the mutations produce an extended ppGpp-dependent pausing of RNA polymerase during the transcription of unstable RNA. Comparison of the vivo and in vitro effects of ppGpp on rrn transcription further suggests that these reflect different phenomena, although in both cases ppGpp inhibits rrn transcription.
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PMID:Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response. 246 Jul 32

A human genomic DNA clone hybridizing to mammalian valine tRNA(IAC) contained a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNA(CUU) gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region. At least nine Alu family members were interspersed throughout the 18.5-kb human DNA fragment, with three Alu elements in the intergenic region between the valine tRNA(AAC) gene and the lysine tRNA gene. Each of the five Alu family members in the sequenced region can be categorized into one of the four Alu subfamilies. The coding regions of all three tRNA genes contain characteristic internal split promoter sequences and typical RNA polymerase III termination signals in the 3'-flanking regions. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase T1 fingerprints of the mature-sized tRNA transcription products are consistent with the structural genes. The lysine tRNA(CUU) gene was transcribed only slightly more efficiently than the valine tRNA(CAC) gene in the homologous in vitro transcription system. Surprisingly, the valine tRNA(CAC) gene was transcribed about eightfold more efficiently than the valine tRNA(AAC) gene, implicating the presence of a modulatory element in the upstream region flanking the tRNA(CAC) gene.
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PMID:A human tRNA gene cluster encoding the major and minor valine tRNAs and a lysine tRNA. 276 31

A library of low Cot DNA (Cot is the molar concentration of DNA times the incubation time in seconds) from Bombyx mori was used to isolate five independent clones of highly reiterated sequences from the genome of this organism. Sequence analysis revealed that all five clones belong to a single family of repetitive DNA elements, which we have named Bm1, and whose reiteration frequency is approximately 2.3 X 10(4) copies per haploid genome. Probing of a Bombyx genomic library (in lambda phage) with a Bm1 clone reveals that this repetitive sequence is dispersed throughout the genome. The pattern of interspersion was confirmed by Southern blot mapping of a large (270 X 10(3) base-pairs) domain of the chorion locus of Bombyx, where at least 13 independent regions were found to hybridize to Bm1. Four additional Bm1 elements have been sequenced from a 4.8 X 10(3) base-pair genomic fragment containing an early chorion gene. Two of these four elements are bounded by short (4 to 12 base-pairs) direct repeats. The nine Bm1 elements which have been sequenced are greater than 88% homologous to each other, and tend to fall in at least two size classes (253 base-pairs and 450 base-pairs). Seven of the nine Bm1 elements have a short 6 to 10 base-pair oligo(A) sequence at the 3' end. A sequence of about 29 base-pairs at the 3' end, including the oligo(A), shows 86% homology to the equivalent 3'-terminal domain of human Alu family repetitive elements. A 129 base-pair domain at the 5' end of Bm1 shows 66% homology to a Drosophila valine transfer RNA gene; thus the 5' end of Bm1 may contain the split internal RNA polymerase III promoter that is characteristic of most transcribed tRNA-like retroposons. Dot-blot analysis of Bombyx RNA shows that Bm1 DNA is indeed transcribed, and that the transcripts are well-represented in the total RNA of an ovarian-derived permanent cell line and posterior silk glands early in the fifth instar, but are less abundant in the RNA of pupae or silk glands late in the fifth instar.
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PMID:A highly reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Bombyx mori. 301 89

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.
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PMID:Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. 306 77

The synthesis of ilv-specific messenger ribonucleic acid (mRNA) by extracts of Escherichia coli K-12 has been demonstrated in a deoxyribonucleic acid (DNA)-dependent, coupled transcription-translation system. ilv-Specific mRNA was determined by hybridization either to double-stranded lambdacI857St68h80dilv DNA (lambdah80dilv DNA) immobilized on nitrocellulose filters or to its separate l and r strands in liquid. During conditions optimal for protein synthesis, slightly more than 6% of the total [(3)H]RNA synthesized by S-30 extracts of the threonine deaminase-negative strain CU5136 was ilv-specific. Of this RNA, nearly 30% was complementary to the l (correct) strand. Total ilv-specific mRNA synthesis in vitro was not affected by omission of valine or all 20 amino acids from the reaction mixture. Hybridization of ilv-specific mRNA made in vitro to the l strand of lambdah80dilv DNA was effectively reduced in the presence of unlabeled RNA extracted from an ilv derepressed strain but not from an ilv deletion strain. In a purified transcription system, employing commercial RNA polymerase, twofold more ilv-specific mRNA was synthesized than in the coupled system, but this increase was entirely due to greater transcription of the r (incorrect) strand. An S-30 extract prepared from a strain isogenic to strain CU5136 but derepressed for ilvA gene expression synthesized twofold more ilv-specific mRNA in the coupled system. The significance of these findings is discussed.
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PMID:Deoxyribonucleic acid-directed in vitro synthesis of ilv-specific messenger ribonucleic acid by extracts of Escherichia coli K-12. 461 11

The production by Neurospora of the enzymes of isoleucine and valine synthesis in response to specific end product-derived signals depends upon the presence of an effective leu-3 regulatory product and its effector alpha-isopropylmalate (alpha-IPM). In leu-3(+) strains, threonine deaminase production is repressed as a function of available isoleucine, acetohydroxy acid synthetase as a function of valine, and the isomeroreductase and dihydroxy acid dehydratase as a function of isoleucine and leucine. In the absence of an effective leu-3 regulatory product, alpha-isopropylmalate, or both, the production of isoleucine and valine biosynthetic enzymes is fixed at or near fully repressed levels even under conditions of severe end product limitation. Thus, in addition to its involvement in the regulation of expression of the three structural genes of leucine synthesis, the leu-3 alpha-IPM regulatory product is necessary for full expression of at least four genes specifying the structure of the enzymes of isoleucine and valine synthesis. It is suggested that the leu-3 alpha-IPM regulatory element may facilitate transcription of the genetically dispersed cistrons either by imposing specificity on ribonucleic acid polymerase for structurally similar promoters adjacent to each of the cistrons or by "opening" promoters after interaction with nearly identical stretches of deoxyribonucleic acid near each of the structural genes.
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PMID:Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora. 482 4


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