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 phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have recently shown that the P protein of VSV, New Jersey serotype (PNJ), expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present work, we are studying the role of phosphorylation in the activation of the P protein of Indiana serotype (PIND), which is highly nonhomologous in amino acid sequence yet structurally similar to its New Jersey counterpart. Despite the fact that E. coli-expressed PIND required phosphorylation by CKII for activation, the phosphorylation negative P protein mutants generated by altering the phosphate acceptors S and T to alanine, surprisingly, showed transcription activity similar to wild-type in vitro. Alteration of S and T residues to phenylalanine, similarly, supported substantial transcription activity (approx. 60% of wild-type), whereas substitution with arginine residue abrogated transcription (approx. 5% of wild-type). In contrast, the same mutants, when expressed in eucaryotic cells, exhibited greatly reduced transcription activity in vitro. This disparate display of transcription phenotype by the PIND mutants expressed in bacteria and eucaryotic cells suggests that these mutants are unique in assuming different secondary structure or conformation when synthesized in two different cellular milieu. The findings that, unless phosphorylated by CKII, the bacterially expressed unphosphorylated (P0) form of PIND, as well as the phosphorylation negative mutants expressed in eucaryotic cells, demonstrates transcription negative phenotype indicate that, like PNJ, phosphorylation of PIND is essential for its activity.
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PMID:Display of disparate transcription phenotype by the phosphorylation negative P protein mutants of vesicular stomatitis virus, Indiana serotype, expressed in E. coli and eucaryotic cells. 936 99

A field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita-MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK2) cells. MVC11 was adapted to LLC-MK2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8 x 10(5) cell-infecting units (CIU) per mouse. On the other hand, with an inoculum of only 4 x 10(1) CIU per mouse, corresponding to 1 LD50, M1 replicated well in mouse lung and was highly virulent to mice. Nucleotide and deduced amino acid sequence analyses of the entire genomes of M1 and MVC11 revealed that adaptation to LLC-MK2 cells and the attenuation of mouse pathogenicity of MVC11 were associated with only two amino acid substitutions; one on the C protein (Phe substituted by Ser at position 170) and the other on the RNA polymerase, the L protein (Glu substituted by Ala at position 2050).
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PMID:Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells. 940 Sep 71

A novel nucleolar component has been identified and cloned using a human autoimmune serum. This antigen, as inferred from the cDNA sequence, is an Mr 55000 protein. Immuno blot analysis, however, of both the native protein and the in vitro translation products of the cDNA showed that they migrate on SDS-PAGE at an apparent molecular mass of 90000 A BLAST search using the cDNA sequence indicated that it is in an antisense orientation to and overlaps the gene of the DNA repair enzyme ERCC-1. An open reading frame, without a translational start site, had been observed by others in this region of the chromosome 19 (19q13.3) and the putative protein was termed ASE-1 (Anti-Sense to ERCC-1). Our cDNA is a full-length equivalent of that open reading frame. ASE-1 was found to contain two domains that are present in a number of nucleolar specific proteins originating from a variety of organisms: a glycine-, arginine- and phenylalanine-rich putative nucleotide interaction domain and an alternating basic/acidic region. Indirect immunofluorescence analysis using antibodies generated to cloned regions of ASE-1 indicated that this protein occurs at the fibrillar centres of the nucleolus in interphase, the putative sites of rDNA transcription, and during cell division it is localized to the nucleolus organizer regions of the chromosomes. ASE-1 co-localises with the RNA polymerase I transcription initiation factor UBF/NOR-90 throughout all stages of the cell cycle and these two proteins associate with each other in vitro.
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PMID:ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes. 942 81

The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function. Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes. The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.
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PMID:DNA-binding domain mutants of sigma-N (sigmaN, sigma54) defective between closed and stable open promoter complex formation. 942 96

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.
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PMID:Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. 946 67

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.
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PMID:Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase. 949 91

Transfer RNA (guanosine-2'-)-methyltransferase (Gm-methylase, EC.2.1.1.32) from extreme thermophile, Thermus thermophilus HB27 is one of the tRNA-ribose modification enzymes; this enzyme specifically catalyze the transfer of a methyl group from S-adenosyl-L-methionine to 2'-OH of the ribose of the guanosine at position 18 in tRNA. A broad substrate specificity of Gm-methylase was observed using natural tRNAs as methyl group acceptors, which suggests that some local stractures common in tRNAs are recognized by the enzyme. By using yeast tRNA(Phe) variants obtained by transcription of their genes with T7 RNA polymerase, it was revealed that the residues G18 and G19, as well as the D-stem structure were primarily required for the methylation reaction and that the essentially minimal sequence for the substrate was Pyrimidine17-G18-G19. The other conserved sequences and the tertiary base-pairs were not essential, but G15, G46, U55 and C56 strongly affected the methylation efficiency.
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PMID:Essentially minimal sequence for substrate recognition by tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27. 958 63

When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3'-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3'-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing. "
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PMID:Recognition sites of 3'-OH group by T7 RNA polymerase and its application to transcriptional sequencing. 960 29

Reverse transcriptase (RT) plays a critical role in retrovirus replication, directing the synthesis of a double- stranded DNA copy of the viral RNA genome. We have previously described a mutant RT of the Moloney murine leukemia virus in which F155 was replaced by valine, and we demonstrated that this substitution allowed the enzyme to incorporate ribonucleotides to form RNA while still retaining its normal ability to incorporate deoxyribonucleotides to form DNA. When introduced into the viral genome, this mutation rendered the virus incapable of replication. Characterization of the mutant virus revealed that the enzyme was still active and able to synthesize minus-strand strong stop DNA and some longer products but failed to make full-length minus-strand DNA. We propose that the failure of the enzyme to complete DNA synthesis in vivo resulted from its ability to incorporate ribonucleotides into the products, which served as inhibitors for DNA synthesis. We also tested seven other amino acid residues for their abilities to substitute for F155 in virus replication; of these, only tyrosine could support virus replication. In an attempt to select for second-site suppressor mutations, the F155V mutant was subjected to random mutagenesis and was used as a parent for the isolation of revertant viruses. Two independent revertants were found to have changed the valine residue at position 155 back to the wild- type phenylalanine. These results suggest that an aromatic ring at this position is important for virus replication.
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PMID:Replication defect of moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides. 962 Oct 52

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.
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PMID:Demonstration that the TyrR protein and RNA polymerase complex formed at the divergent P3 promoter inhibits binding of RNA polymerase to the major promoter, P1, of the aroP gene of Escherichia coli. 976 83


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