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)

Ten aminoacyl transfer RNA's prepared from human malignant trophoblastic cells (BeWo line) were compared with the corresponding aminoacyl transfer RNA's from normal human chorionic tissue by cochromatography on a RPC-5 column. Phenylalanyl transfer RNA (Phe-tRNA) of BeWo cells had, in addition to the single species of Phe-tRNA found in normal chorionic tissues, an early eluting component. When Phe-tRNA from the chorion was exposed to mild acid, which selectively excises the Y base, it eluted in the same position as the early eluting Phe-tRNA of BeWo cells. Therefore, the BeWo Phe-tRNA is partially undermodified. Tyrosyl transfer RNA of BeWo cells exhibited a broad-based peak which eluted later than the normal and probably consists of two or more tyrosyl transfer RNA's. Seryl transfer RNA of BeWo cells showed two peaks of acceptor activity, while seryl transfer RNA of normal chorion had a third peak that eluted at a higher salt concentration. In addition, in an early eluting methionyl and lysyl transfer RNA and in a late eluting arginyl transfer RNA from BeWo cells and normal charion, quantitative alterations were detected. The remaining four transfer RNA's, leucyl, aspartyl, valyl, and histidyl, from the two sources did not show any significant differences in elution profiles. These alterations of the chromatographic profile appeared to be due to new or altered species of transfer RNA. They were not due to differences in the aminoacyl transfer RNA synthetase. The transfer RNA methyltransferase capacity of the enzymes from BeWo cells was 2-fold higher than that of the enzymes extracted from the chorion.
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PMID:Changes in transfer RNA's in human malignant trophoblastic cells (BeWo line). 17 10

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.
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PMID:Two methyltransferase activities in the purified virions of vesicular stomatitis virus. 20 77

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.
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PMID:Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. 151 55

Computer-assisted comparisons of the large proteins involved in the replication of viral RNA have revealed a novel domain located near the N termini of these proteins and conserved throughout the so-called 'Sindbis-like' supergroup of positive-strand RNA viruses. This domain encompasses four distinct conserved motifs, with motifs I, II and IV containing an invariant His residue, the AspXXArg signature and an invariant Tyr residue, respectively. Each of the two large groups of viruses within this supergroup, the 'altovirus' group (alphaviruses, tobamoviruses, tobraviruses, hordeiviruses, tricornaviruses, furoviruses, hepatitis E virus and probably rubiviruses), and the 'typovirus' group (tymoviruses, potexviruses, carlaviruses and apple chlorotic leaf spot virus), can be characterized by additional conserved sequence motifs. Based on the available results of biochemical studies and site-directed mutagenesis of the alphavirus proteins, it is hypothesized that this domain may be involved in methylation of the cap during viral RNA maturation. Unlike the other conserved domains, the RNA-dependent RNA polymerase and the RNA helicase, the motifs typical of the putative methyltransferase domain are universal within the Sindbis-like supergroup but are not found in the proteins of any other viruses, constituting a distinctive hallmark of this supergroup.
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PMID:Conservation of the putative methyltransferase domain: a hallmark of the 'Sindbis-like' supergroup of positive-strand RNA viruses. 164 51

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV 1a and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the 1a and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the 1a protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the 1a and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.
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PMID:Localization of functional regions of the cucumber mosaic virus RNA replicase using monoclonal and polyclonal antibodies. 752 62

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Complete sequence of the citrus tristeza virus RNA genome. 774 24

The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.
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PMID:Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease. 825 66

A sequence motif that is conserved in a number of S-adenosylmethionine (SAM)-utilizing methyltransferases and is implicated in SAM binding was identified in the N-terminal portion of NS5 proteins of flaviviruses and in lambda 2 protein of reovirus. An additional conserved motif was shared by these viral proteins and two distinct groups of methyltransferases including as the prototypes Rhodobacter capsulatus hydroxyneurosporene methylase (crtF gene product) and yeast 3,4-dihydroxy-5-hexaprenylbenzoate methylase (COQ3 gene product), respectively. Statistically significant similarity was revealed between the region of flavivirus NS5 containing the SAM-binding motif and a newly characterized family of putative methyltransferases from bacteria, yeast and plants, which is related to the Coq3 group. Amino acid sequence signatures were derived that are unique for NS5 proteins and different subsets of (putative) cellular methyltransferases. It is hypothesized that the N-terminal domain of NS5 is a methyltransferase involved in viral RNA capping. Thus NS5 may be a two-domain protein, with its C-terminal domain comprising the RNA-dependent RNA polymerase. The putative methyltransferase domain of flaviviruses is unrelated to the methyltransferase domain previously characterized in positive-strand RNA viruses of the alphavirus-like supergroup. The lack of sequence similarity and different location of the putative methyltransferase domain underscores the drastic difference in the genome layout of flaviviruses and alphaviruses. The identification of the putative methyltransferase domain in reovirus lambda 2 protein is compatible with the available evidence that this protein is the viral capping enzyme.
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PMID:Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. 838 98

Grapevine virus B (GVB) is a tentative member of the genus Trichovirus. The 5'-terminal region of the RNA genome of GVB comprises 5437 nucleotides and has been sequenced by the dideoxynucleotide chain termination method. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORFs) were identified. ORF 1 coded for a 194.7 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses (i.e. methyltransferase, helicase and RNA-dependent RNA polymerase, in that order from the N to the C terminus). ORF 2 encoded a 20 kDa polypeptide that did not show any significant sequence homology with protein sequences from the databases. The biological function of this polypeptide was not determined. Although the 20 kDa product was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and an antiserum produced, it could not be identified in GVB-infected plant tissue extracts. The GVB genome had the same size as that of apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, but differed substantially in the number (five compared to three), size and order of genes. Differences existed also in the extent of sequence homology between polymerases, which did not cluster together in tentative phylogenetic trees. The results of this study show that definitive and tentative trichovirus species differ molecularly to an extent that may warrant a taxonomic revision of the genus.
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PMID:The nucleotide sequence and genomic organization of grapevine virus B. 888 2


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