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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass, M. G. Mulkerrin, and J. A. Wells, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paoni, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. Zoller, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragenic suppression analysis of such mutant viruses may be very useful in identifying components of the viral replication complex.
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PMID:Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis. 828 89

Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins. In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions. Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution. Besides the amino and the carboxyl groups there is no functional group in the molecule. Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain'. Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2). The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme). The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies. In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases. In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions. Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem. The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase. Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced. They encode synthetase subunits of chain lengths ranging from 300-700 amino acids. One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined. The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly. Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease.
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PMID:Glycyl-tRNA synthetase. 883 80

Activated human and rat T cells as well as mouse T-cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II+ antigen (Ag) presenting T cells to induce proliferation of Ag-specific cloned T cells has been controversial. We analysed whether the failure of some T-cell clones to proliferate in response to Ag presented by class II+ T cells is because of a lack of costimulatory cytokine production by the antigen-presenting cells (APC). As a model system the mouse class II+ cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T-helper 1 (Th1) type, GAT (synthetic copolymer of L-glutamic acid, L-alanine and L-tyrosine)-specific clone is characterized by an efficient downregulation of interleukin-2 receptor (IL-2R) with time following antigenic stimulation. KIII5 cells respond to GAT-presenting splenic antigen-presenting cells (APC) by IL-2 production, IL-2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL-2, but did not proliferate. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a lack of IL-12 production by BI/O4.1 cells. Addition of IL-12 to a coculture of Ag-presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL-12 in synergy with IL-2 upregulated IL-2R alpha chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II+ T cells are not functional in inducing Ag-mediated expansion of resting Th1 cells owing to their failure to produce IL-12, but rather that they play a role in amplification loops during an ongoing immune response.
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PMID:Costimulatory signalling potential of murine MHC class II-positive T-clone cells. 895 51

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.
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PMID:Detection of mutant p53 in clam leukemia cells. 916 98

We have analyzed one of the functional domains of Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta. Deletion mapping analysis of the carboxyl-terminal region of the beta-subunit protein revealed that the terminal 18 amino acid residues (positions 571-588) are dispensable for the replicase reaction. Subsequent deletions up to the Ala-565 residue reduced the RNA polymerizing activity of the replicase in vivo but increased it in vitro. The mutant replicases with enhanced in vitro RNA polymerizing activity were found to have relaxed template specificity for ribosomal RNAs and cellular RNAs as well as Qbeta RNA. Deletions beyond the Ile-564 residue abolished both the RNA polymerizing activity and the binding ability to midivariant (MDV)-poly(+) RNA, a derivative of a natural template for Qbeta replicase, MDV-1 RNA. These results suggest that the carboxyl-terminal part of the beta-subunit participates in RNA recognition of Qbeta replicase.
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PMID:Deletion analysis of Qbeta replicase. Participation of the carboxyl-terminal region of the beta-subunit protein in template recognition. 918 62

The RNA-dependent RNA polymerase (RdRp) of potato virus X (PVX) contains a glycine-lysine-serine (GKS) motif. This motif is present in the replication enzyme of many RNA viruses and is thought to be required for nucleoside triphosphate-binding. Three single amino acid changes, glycine to alanine (AKS), lysine to asparagine (GNS) and lysine to glutamate (GES) within the GKS motif of the PVX RdRp were tested for their effect on PVX accumulation. The GNS and GES mutations rendered the virus unable to accumulate in either tobacco plants or protoplasts, whereas substitution of glycine with alanine had only a minor effect on accumulation of PVX. The glycine to alanine mutation reverted to wild-type after passage on Nicotiana clevelandii plants. These findings suggest that the GKS motif is required for PVX replication and that strong selection pressures are active to maintain necessary sequences of the viral RdRp.
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PMID:Mutation of the GKS motif of the RNA-dependent RNA polymerase from potato virus X disables or eliminates virus replication. 919 15

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels > or = 70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P(60/62/64)) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. Increasing the amount of P(60/62/64) expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P(TTT)) had no apparent effect on transcription (113% of the wild-type level) or replication (81% of the wild-type level), substitution with phenylalanine (P(FFF)) rendered the protein much less active in transcription (< 5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P(EEE)) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P(RRR)) or transcription (P(60/62/64)) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L.
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PMID:Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. 934 67

The phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein N in vivo. We have shown that the deletion of 11 amino acids from the C terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesis in vitro in a transcription reconstitution reaction as well as transcription in vivo of a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) both in vitro and in vivo, suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N protein in vivo, indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNA in vivo. Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively.
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PMID:Basic amino acid residues at the carboxy-terminal eleven amino acid region of the phosphoprotein (P) are required for transcription but not for replication of vesicular stomatitis virus genome RNA. 937 14

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.
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PMID:Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3Dpol with VPg and with genetic variants of 3AB. 965 21

Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.
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PMID:Design of selective eglin inhibitors of HCV NS3 proteinase. 970 81


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