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)

Three new rif-r-mutations, obtained independently, were localized in the rpoB gene coding for the beta-subunit of DNA-dependent RNA polymerase of E. coli. Two of them led to identical Asp(516)-Asn amino acid substitution with relatively low resistance of corresponding E. coli strains to rifampicin. The third mutation affected the His 526 residue transforming it into Tyr and endowed the E. coli cells with a high resistance against rifampicin.
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PMID:[Nucleotide substitutions in the rpoB gene leading to rifampicin resistance of E. coli RNA polymerase]. 638 89

Purified calf thymus RNA polymerase II is composed primarily of species IIA and IIB. These enzymes differ in the apparent molecular weight of their largest subunit, designated IIa and IIb for enzyme forms IIA and IIB, respectively. Both enzyme forms contain an additional high molecular weight subunit designated IIc. The structural relationship between subunits IIa, IIb, and IIc, labeled with 125I under both native and denaturing conditions, has been analyzed by two-dimensional peptide mapping. Native RNA polymerase II was iodinated and subunits IIa, IIb, and IIc purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The subunits were then digested with either trypsin or thermolysin and the 125I-labeled peptides resolved by thin layer electrophoresis in the first dimension and chromatography in the second dimension. Similar peptide maps were obtained for each of the three large subunits, suggesting that subunits IIa, IIb, and IIc are related in primary sequence. Alternatively, RNA polymerase subunits IIa, IIb, and IIc were purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, eluted from the gel, and then iodinated. The use of denatured subunits as substrate for the iodination eliminates the differential reactivity of specific tyrosine residues imposed by the structure of the native protein. Under these labeling conditions, the tryptic and thermolytic peptide maps of subunits IIa and IIb are nearly identical but bear much less resemblance to the peptide maps of subunit IIc than with the previous labeling procedure. These results suggest that subunits IIa and IIb are closely related in primary sequence but cannot establish whether these subunits are the products of closely related genes or are related by processing at the level of primary transcript or primary translation product. Subunit IIc bears a more distant relationship to subunits IIa and IIb. Possible reasons why this homology is only apparent in peptide maps from subunits labeled in the native enzyme are discussed.
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PMID:Structural relationship between the large subunits of calf thymus RNA polymerase II. 683 37

E. coli ribosomal protein L12, because of its unique features, has been studied in more detail than perhaps any of the other ribosomal proteins. Unlike the other ribosomal proteins that are generally present in stoichiometric amounts, there are four copies of L12 per ribosome, some of which are acetylated on the N-terminal serine. The acetylated species, referred to as L7, has not been shown, as yet, to possess any different biological activity than L12. A specific enzyme that acetylates L12 to form L7, using acetyl-CoA as the acetyl donor, has been purified from E. coli extracts. L12 is also unique in that it does not contain cysteine, tryptophan, histidine, or tyrosine, is very acidic (pI: 4.85) and has a high content of ordered secondary structure (approximately 50%). The protein is normally found in solution as a dimer and also forms a tight complex with ribosomal protein L10. There are three methionine residues in L12, located in the N-terminal region of the protein, one or more of which are essential for biological activity. Oxidation of the methionines to methionine sulfoxide prevents dimer formation and inactivates the protein. The four copies of L12 are located in the crest region(s) of the 50S ribosomal subunit. There is good evidence that the soluble factors, such as IF-2, EF-Tu, EF-G and RF, interact with L12 on the ribosome during the process of protein synthesis. This interaction is essential for the proper functioning of each of the factors and for GTP hydrolysis associated with the individual partial reactions of protein synthesis. The L12 gene is located on an operon that contains the genes for L10 and beta beta' subunits of RNA polymerase at about 88 min on the bacterial chromosome. DNA-directed in vitro systems have been used to study the unique regulation of the expression of these genes. Autogenous regulation, translational control, and transcription attenuation are regulatory mechanisms that function to control the synthesis of these proteins.
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PMID:Chemistry and biology of E. coli ribosomal protein L12. 701 80

The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme. Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C. D. Morrow, J. Virol. 67:373-381, 1993). The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase. On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase. To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326). The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed. Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108. In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type. The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation. Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed. Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus. Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus. The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus. RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.
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PMID:An aspartic acid at amino acid 108 is required to rescue infectious virus after transfection of a poliovirus cDNA containing a CGDD but not SGDD amino acid motif in 3Dpol. 749 45

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Phosphorylation of the CTD occurs during formation of the initiation complex and is correlated with the transition from complex assembly to elongation. Previously, serine and threonine residues within the CTD have been shown to be modified by the addition of phosphate and by the addition of O-linked GlcNAc. Our results establish that the CTD is also modified in vivo by phosphorylation on tyrosine. Furthermore, a nuclear tyrosine kinase encoded by the c-abl protooncogene phosphorylates the CTD to a high stoichiometry in vitro. Under conditions of maximum phosphorylation, approximately 30 mol of phosphate are incorporated per mol of CTD. The observation that the CTD is not phosphorylated by c-Src tyrosine kinase under identical conditions indicates that the CTD is not a substrate of all tyrosine kinases. Phosphorylation of tyrosine residues within the CTD may modulate the interaction of RNA polymerase II with the preinitiation complex and, hence, may be important in regulating gene expression.
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PMID:Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain. 750 85

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.
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PMID:Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors. 750

The molecular cloning of a murine receptor-type protein tyrosine phosphatase, termed PTP NU-3, with an extracellular cell-adhesion-molecule-like domain is reported. NU-3 was isolated from 11.5-day total mouse embryonic RNA by reverse-transcriptase PCR using degenerate oligonucleotides flanking the conserved protein tyrosine phosphatase catalytic domain. This produced a 280-bp DNA probe which was subsequently employed to screen a mouse embryonic kidney library. Several overlapping cDNA clones were isolated, collectively forming a cDNA of 6.0 kb that encodes a putative 211-kDa protein. Northern-blot analysis of total RNA from adult and embryonic mouse tissues indicates the existence of two major PTP NU-3 transcripts of approximately 6 kb and 7 kb. Both messages are expressed predominantly in brain tissues and neuronal-derived cell lines, although detectable levels of the 7-kb message were found in other non-neuronal tissues. We have identified a unique 132-bp exon segment that is present in the 7-kb message but is completely absent in the 6-kb transcript, suggesting tissue-specific levels of expression and RNA processing. Analysis of the amino acid sequence encoded by the 132-bp segment reveals that it completes a partial fibronectin type-III element resulting in a protein with a total of nine such elements. Bacterial expression of the two catalytic domains demonstrated that only the first domain possesses enzymic activity towards a tyrosine phosphorylated substrate.
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PMID:Molecular cloning and tissue-specific RNA processing of a murine receptor-type protein tyrosine phosphatase. 752 77

The Src-homology (SH) 2 domain, found in a variety of proteins, has a binding site for phosphotyrosine-containing peptides. In adaptor proteins such as Grb2, the SH2 domain plays an important role in the assembly of signal transducer complexes. Many nonreceptor tyrosine kinases--e.g., Abl and Src--also contain SH2 domains. Without a functional SH2 domain, these tyrosine kinases retain catalytic activity but lose their biological function. This result suggests that the SH2 domain may be involved in the selection of biologically relevant substrates. We have previously shown that the carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl but not the Src tyrosine kinase. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated [Tyr(P)] CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2 or exchanging it with that of Src, which does not bind the Tyr(P)-CTD, abolished processivity and reduced the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity.
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PMID:Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain. 753 94

Structural and functional characteristics of the goat uterine nuclear estrogen receptor R-II have been subjected to comparison with those of the nonactivated estrogen receptor (naER), purified from the cytosol. The two proteins have the same molecular mass, 66 kDa; they display identical peptide maps and are both recognized by anti-estrogen receptor (R-I) IgG. Both are tyrosine kinases and bind with equal affinity to a column of anti-phosphotyrosine IgG-Sepharose. On the other hand, while naER is a glycoprotein, the R-II does not show any sign of glycosylation. Unlike the naER, the R-II is incapable of dimerization with estrogen receptor activation factor (E-RAF) and, as a consequence, bind to the DNA. R-II has a higher estradiol binding capacity and the resultant reduction in its affinity for the hormone in comparison with the naER. Further, the sedimentation behavior and the Stokes radius of the naER indicate a globular nature in the shape of the protein. The corresponding data for the R-II reveal that the protein has a distinct nonglobular shape. Deglycosylation of the naER using a glycopeptidase resulted in the total conversion of the distinct physical features of the naER to the R-II category. This treatment resulted, without effecting any reduction in its molecular mass, in the loss of the E-RAF dimerization capacity of the naER. The Stokes radius and the sedimentation coefficient of the protein underwent drastic changes and became closely similar to those of the R-II. In addition, the deglycosylation introduced a several-fold enhancement in the capacity of the naER to bind estradiol with a concomitant decrease in its affinity, similar to the corresponding properties of the R-II. The R-II is shown to have a conformational structure different from that of the naER, to interact with the nuclear RNA polymerase II. It is also shown here that the R-II phosphorylates two subunits (molecular mass 91 and 20 kDa) in the RNA polymerase II, in addition to the 40-kDa subunit phosphorylated by the naER. The results clearly indicate the possibility that the nuclear R-II estrogen receptor is the deglycosylated naER.
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PMID:The nuclear estrogen receptor R-II of the goat uterus: distinct possibility that the R-II is the deglycosylated form of the nonactivated estrogen receptor (naER). 754 97

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.
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PMID:A mutant T7 RNA polymerase as a DNA polymerase. 755 4


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