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)

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.
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PMID:Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase. 238 51

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.
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PMID:Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei. 852 30

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

Double-stranded RNA (dsRNA) viruses are complex RNA processing machines that sequentially perform packaging, replication and transcription of their genomes. In order to characterize the assembly intermediates of such a machine we have developed an efficient in vitro assembly system for the procapsid of bacteriophage phi8. The major structural protein P1 is a stable and soluble tetramer. Three tetramers associate with a P2 monomer (RNA-dependent RNA polymerase) to form the nucleation complex. This complex is further stabilized by a P4 hexamer (packaging motor). Further assembly proceeds via rapid addition of individual building blocks. The incorporation of the packaging and replication machinery is under kinetic control. The in vitro assembled procapsids perform packaging, replication and transcription of viral RNA. Comparison with another dsRNA phage, phi6, indicates conservation of key assembly intermediates in the absence of sequence homology and suggests that a general assembly mechanism for the dsRNA virus lineage may exist.
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PMID:Conserved intermediates on the assembly pathway of double-stranded RNA bacteriophages. 1272 55

Bacteriophage Phi2954 contains three dsRNA genomic segments, designated L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The core particle is covered by a shell of protein P8, and this structure is enclosed within a lipid-containing membrane. We have found that normal infection of the host Pseudomonas syringae is dependent on the action of a host protein, glutaredoxin 3 (GrxC). GrxC removes the P8 shell from the infecting particle and binds to the inner core. Removal of P8 activates the transcription of segments S and M, whereas binding of GrxC to the core particle activates the transcription of segment L. The differences in transcription behavior are due to the preference of the polymerase for G as the first base of the transcript. Transcripts of segments S and M begin with GCAA, whereas those of segment L begin with ACAA. The binding of GrxC to the particle results in changes in polymerase activity. Mutations resulting in independence of GrxC are found in the gene for protein P1, the major structural protein of the inner core particle.
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PMID:Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection. 2023 37

All representatives of rhabdoviruses contain a nucleocapside phosphoprotein - P-protein which is an essential subunit of the viral RNA-dependent RNA polymerase complex. As a result of studying the effect of nucleocapside protein P(NS) on replicase activity of mRNP isolated from plants infected by potato curly dwarf virus in the system in vitro, it was established that nucleocapside P-protein stimulates considerably the replicase activity of membrane-bound polysomal m-RNP P-protein being available in concentration of 15 microg/ml in the replication system in vitro of membrane-bound polysomal mRNP, the replicase activity increased 11.7 times. This property of nucleocapside P-protein at the same concentration was displayed to a less extent with the presence of free polysomal mRNP, in the system in vitro. Thus the replicase activity mRNP-complexes in the replication system in vitro depends on the presence of nucleocapside viral P-protein in the system. Its concentration being increased or decreased, one can observe the change of the replicase activity.
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PMID:[Effect of structural P-protein of potato curly dwarf virus on replicase activity of mRNP-complexes of infected plants]. 2230 54

Potyviruses (family Potyviridae, genus Potyvirus) are the result of an initial radiation event that occurred 6,600 years ago. The genus currently consists of 167 species that infect monocots or dicots, including domesticated and wild plants. Potyviruses are transmitted in a non-persistent way by more than 200 species of aphids. As indicated by their wide host range, worldwide distribution, and diversity of their vectors, potyviruses have an outstanding capacity to adapt to new hosts and environments. However, factors that confer adaptability are poorly understood. Viral RNA-dependent RNA polymerases introduce nucleotide substitutions that generate genetic diversity. We hypothesized that selection imposed by hosts and vectors creates a footprint in areas of the genome involved in host adaptation. Here, we profiled genomic and polyprotein variation in all species in the genus Potyvirus. Results showed that the potyviral genome is under strong negative selection. Accordingly, the genome and polyprotein sequence are remarkably stable. However, nucleotide and amino acid substitutions across the potyviral genome are not randomly distributed and are not determined by codon usage. Instead, substitutions preferentially accumulate in hypervariable areas at homologous locations across potyviruses. At a frequency that is higher than that of the rest of the genome, hypervariable areas accumulate non-synonymous nucleotide substitutions and sites under positive selection. Our results show, for the first time, that there is correlation between host range and the frequency of sites under positive selection. Hypervariable areas map to the N terminal part of protein P1, N and C terminal parts of helper component proteinase (HC-Pro), the C terminal part of protein P3, VPg, the C terminal part of NIb (RNA-dependent RNA polymerase), and the N terminal part of the coat protein (CP). Additionally, a hypervariable area at the NIb-CP junction showed that there is variability in the sequence of the NIa protease cleavage sites. Structural alignment showed that the hypervariable area in the CP maps to the N terminal flexible loop and includes the motif required for aphid transmission. Collectively, results described here show that potyviruses contain fixed hypervariable areas in key parts of the genome which provide mutational robustness and are potentially involved in host adaptation.
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PMID:Genome-Wide Variation in Potyviruses. 3179 6

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