Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.
 ...
PMID:Solubilization and promoter analysis of RNA polymerase from rice stripe virus. 152 54

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
 ...
PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3' viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5' noncoding region and very poorly by the 3' conserved 98-nucleotide tail. A 3' coding region with conserved stem-loop structures rather than the 3' noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3' coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.
 ...
PMID:Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA. 1040 Aug 7

Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.
 ...
PMID:Analysis of cis-acting sequences involved in plus-strand synthesis of a turnip crinkle virus-associated satellite RNA identifies a new carmovirus replication element. 1070 42

The polyoxin (POL) biosynthetic gene cluster (pol) was recently cloned from Streptomyces cacaoi subsp. asoensis. A 3.3 kb DNA fragment carrying an obvious open reading frame (polR), whose deduced product shows sequence similarity to SanG of Streptomyces ansochromogenes and PimR of Streptomyces natalensis, was revealed within the pol gene cluster. Disruption of polR abolished POL production, which could be complemented by the integration of a single copy of polR into the chromosome of the non-producing mutant. The introduction of an extra copy of polR in the wild-type strain resulted in increased production of POLs. The transcription start point (tsp) of polR was determined by S1 mapping. Reverse transcriptase PCR experiments showed that PolR is required for the transcription of 18 structural genes in the pol gene cluster. Furthermore, we showed that polC and polB, the respective first genes of two putative operons (polC-polQ2 and polA-polB) consisting of 16 and 2 of these 18 genes, have similar promoter structures. Gel retardation assays indicated that PolR has specific DNA-binding activity for the promoter regions of polC and polB. Our data suggest that PolR acts in a positive manner to regulate POL production by activating the transcription of at least two putative operons in the pol gene cluster.
 ...
PMID:polR, a pathway-specific transcriptional regulatory gene, positively controls polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis. 1938 87

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3' translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3' end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3' end, which causes structural changes that potentiate the shift between translation and replication.
 ...
PMID:The 3' end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase. 1965 66

There are two protein primers involved in picornavirus RNA replication, VPg, the viral protein of the genome, and VPgpUpU(OH). A cis-acting replication element (CRE) within the open reading frame of poliovirus (PV) RNA allows the viral RNA-dependent RNA polymerase 3D(Pol) to catalyze the conversion of VPg into VPgpUpU(OH). In this study, we used preinitiation RNA replication complexes (PIRCs) to determine when CRE-dependent VPg uridylylation occurs relative to the sequential synthesis of negative- and positive-strand RNA. Guanidine HCl (2 mM), a reversible inhibitor of PV 2C(ATPase), prevented CRE-dependent VPgpUpU(OH) synthesis and the initiation of negative-strand RNA synthesis. VPgpUpU(OH) and nascent negative-strand RNA molecules were synthesized coincident in time following the removal of guanidine, consistent with PV RNA functioning simultaneously as a template for CRE-dependent VPgpUpU(OH) synthesis and negative-strand RNA synthesis. The amounts of [(32)P]UMP incorporated into VPgpUpU(OH) and negative-strand RNA products indicated that 100 to 400 VPgpUpU(OH) molecules were made coincident in time with each negative-strand RNA. 3'-dCTP inhibited the elongation of nascent negative-strand RNAs without affecting CRE-dependent VPg uridylylation. A 3' nontranslated region mutation which inhibited negative-strand RNA synthesis did not inhibit CRE-dependent VPg uridylylation. Together, the data implicate 2C(ATPase) in the mechanisms whereby PV RNA functions as a template for reiterative CRE-dependent VPg uridylylation before and during negative-strand RNA synthesis.
 ...
PMID:Conversion of VPg into VPgpUpUOH before and during poliovirus negative-strand RNA synthesis. 1981 61

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.
 ...
PMID:The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis. 2044 70