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)

The L-A double-stranded RNA virus of Saccharomyces cerevisiae makes a gag-pol fusion protein by a -1 ribosomal frameshift. The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants. We have carried out "alanine-scanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N (. = any amino acid) and GDD. By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely defined the extent of domains and specific residues essential for viral replication. Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely beta-sheet structure.
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PMID:RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains. 154 80

A region of the feline calicivirus (FCV) genome was sequenced which encodes polypeptides that are similar by amino acid alignment analysis to several picornavirus polypeptides. These polypeptides include the 2C polypeptide, the 3C cysteine protease and the 3D RNA-dependent RNA polymerase. The 2C-like region of the FCV genome encodes a GxxGxGKT nucleotide binding motif as well as amino acids which have been shown to be conserved in the picornavirus 2C polypeptides. The FCV RNA-dependent RNA polymerase also shows regions of similarity with picornavirus RNA polymerase sequences including the GDD sequence which is thought to be in or near the active site of the polymerase. The FCV cysteine protease-like sequences have the lowest degree of similarity with picornavirus cysteine proteases of the three regions aligned. However, the cysteine and histidine residues thought to be in the active site of the protease are present and are surrounded by amino acids conserved in the picornavirus cysteine proteases. The order of the polypeptides encoded in the FCV genome is the same as in the picornaviruses with the RNA-dependent RNA polymerase being located at the C-terminus of the FCV polyprotein. However, there is an approximately 40,000 dalton region between the FCV 2C- and the cysteine protease-like polypeptides which has no similarity to any known picornavirus protein. A striking difference between the organization of these sequences in FCV and the picornaviruses is that in the FCV genome, these non-structural polypeptides are encoded near the 5' end of the genomic RNA. Termination of the reading frame encoding these polypeptides occurs approximately 2400 bases from the 3' end of the genomic RNA as compared to 71 bases in the poliovirus genomic RNA.
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PMID:Nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptides. 207 82

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 poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity. 785 86

The two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb-CP interaction but not the NIb-NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb-NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.
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PMID:A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. 852 11

We have expressed the putative RNA-dependent RNA polymerase encoded by the potyvirus tobacco vein mottling virus (TVMV) in Escherichia coli as a glutathione S-transferase fusion protein. As prepared, the fusion protein possessed the poly(U) polymerase activity that is a hallmark of other picornavirus-encoded polymerases. In addition, this protein was able to utilize full-length TVMV RNA as a template for RNA synthesis. A fusion protein containing a mutation in the highly conserved GDD motif of the polymerase (GDD-->ADD) possessed 7% of the activity of the wild type. Our results confirm that the presumed polymerase encoded by TVMV is in fact an RNA-dependent RNA polymerase and that the GDD motif so widely seen in viral polymerases has an important function in the TVMV protein.
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PMID:RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase. 894 34

The NIb protein of tobacco etch potyvirus (TEV) possesses several functions, including RNA-dependent RNA polymerase and nuclear translocation activities. Using a reporter protein fusion strategy, NIb was shown to contain two independent nuclear localization signals (NLS I and NLS II). NLS I was mapped to a sequence within amino acid residues 1 to 17, and NLS II was identified between residues 292 and 316. Clustered point mutations resulting in substitutions of basic residues within the NLSs were shown previously to disrupt nuclear translocation activity. These mutations also abolished TEV RNA amplification when introduced into the viral genome. The amplification defects caused by each NLS mutation were complemented in trans within transgenic cells expressing functional NIb, although the level of complementation detected for each mutant differed significantly. Combined with previous results (X. H. Li and J. C. Carrington, Proc. Natl. Acad. Sci. USA 92:457-461, 1995), these data suggest that the NLSs overlap with essential regions necessary for NIb trans-active function(s). The fact that NIb functions in trans implies that it must interact with one or more other components of the genome replication apparatus. A yeast two-hybrid system was used to investigate physical interactions between NIb and several other TEV replication proteins, including the multifunctional VPg/proteinase NIa and the RNA helicase CI. A specific interaction was detected between NIa and NIb. Deletion of any of five regions spanning the NIb sequence resulted in NIb variants that were unable to interact with NIa. Clustered point mutations affecting the conserved GDD motif or NLS II within the central region of NIb, but not mutations affecting NLS I near the N terminus, reduced or eliminated the interaction. The C-terminal proteinase (Pro) domain of NIa, but not the N-terminal VPg domain, interacted with NIb. The effects of NIb mutations within NLS I, NLS II, and the GDD motif on the interaction between the Pro domain and NIb were identical to the effects of these mutations on the interaction between full-length NIa and NIb. These data are compatible with a model in which NIb is directed to replication complexes through an interaction with the Pro domain of NIa.
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PMID:Functions of the tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa). 899 87

Genome of GBV-C have 5' and 3' untranslated region and a coding region of 2842 amino acid. In comparison with other flaviviruses, GBV-C lacks the sequence coding typical CORE protein at the N terminus of putative polyprotein. In the putative structure region, E1 and E2, three potential glycosylation sites(Asn-X-Ser/Thr) were conserved in all isolates. In the putative NS3 region, conserved elements found in RNA helicases with nucleotide triphosphate(NTP) binding motif, GSGKS(residue 1097-1101) and DECH(residue 1184-1187), were observed. In addition, a sequence motif found in all viral RNA-dependent RNA polymerase, GDD(residue 2587-2589) was also conserved in NS5B region.
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PMID:[Genetic organization of GBV-C]. 908 53

The complete nucleotide sequence of major core protein gene (segment S3) of rice dwarf virus (RDV) Chinese isolate was determined after cDNA cloning from the viral genomic RNA. Sequence analysis showed that the cloned fragment is 3195 bp in length and contains a single open reading frame (ORF), encoding the major core protein (P3) which M(r) of 114 K. The nucleotide and deduced amino acid sequences of S3 of this isolate share significant homology (94.1% and 97%, respectively) with those of S3 of the Japanese isolate. At the amino acid level, P3 of RDV Chinese isolate shares significant homology with P3 of rice gall dwarf virus (RGDV), significant regional homology with the rotavirus penetration, and homology with spheroidin of amsacta entomopoxvirus (SPH), which is the major protein of the occlusion body, with clp-like ATP-dependent protease binding subunit and with ATP-dependent protease ATP-binding subunit. Amino acid sequence analysis also showed that P3 contains RNA-dependent RNA polymerase (RDRP) motif-like elements such as DXXXD, SGXXXXXXN, GDD and ENXXXY. These results may suggest that P3 is a multifunctional protein which plays very important roles in the virus structure formation, virus replication and penetration processes. The full length cDNA sequence of RDV S3 and a partial one which covers nt 1004-3195 were cloned into bacterial expression vector pTrcHisB for expression. The full length cDNA sequence failed to be expressed in E. coli, but the partial sequence was successfully expressed there as confirmed by the Western blot analysis. Further analysis of RDV P3 is under way.
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PMID:Molecular cloning, sequencing, functional analysis and expression in E. coli of major core protein gene (S3) of rice dwarf virus Chinese isolate. 938 5

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.
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PMID:RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. 962 34


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