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)

Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12-14-nucleotide GC-rich RNA duplex between the end of the 5' tRNA half and the beginning of the 3' tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNA(His) and tRNA(Glu) species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.
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PMID:Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. 1569 44

In this study, Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis omega virus (NomegaV). DpTV particles are isometric, with a diameter of about 40 nm and a buoyant density of 1.281 g cm(-3) in CsCl. The virus has two capsid proteins (of 62 500 and 6800 Da) and two single-stranded RNA molecules (RNA1 and RNA2), which are 5492 and 2490 nt long, respectively. RNA1 has a large open reading frame (ORF) encoding a polypeptide of 180 kDa; RNA2 contains two partially overlapping ORFs encoding polypeptides of 17 and 70 kDa. The 180 kDa protein, which contains consensus motifs of a putative methyltransferase, helicase and RNA-dependent RNA polymerase, shows significant similarity to those of other tetraviruses. The 17 kDa protein is a PEST (Pro/Glu/Ser/Thr) protein of unknown function. The 70 kDa protein is the coat protein precursor and is predicted to be cleaved at an Asn-Phe site located after residue 570. The 70 kDa protein shows 86 and 66 % identity to its homologues in NomegaV and Helicoverpa armigera stunt virus, respectively. Secondary-structure analysis revealed that the RNAs of DpTV have tRNA-like structures at their 3' termini.
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PMID:Isolation and identification of a new tetravirus from Dendrolimus punctatus larvae collected from Yunnan Province, China. 1572 41

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
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PMID:ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture. 1618 75

The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). A model for this, based on the core of the large subunit of the ribosome, is developed further. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have "poisoned" the ribopolymerase by cross-reacting with the nucleoside triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of amino-acylated tRNA-like products. Using base-pairing interactions, it is suggested that some of these molecules might cross-link two ribopolymerases giving rise to a precursor of the modern ribosome with two subunits linked by tRNA. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors. Some implications for the genetic code are discussed.
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PMID:A molecular model for the origin of protein translation in an RNA world. 1691 61

The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). If such a replicase makes a reverse complementary copy of any sequence (including itself), in a simple RNA world, there is no mechanism to prevent self-hybridization. It is proposed that this can be avoided through the synthesis of a parallel complementary copy. The logical consequences of this are pursued and developed in a computer simulation, where the behaviour of the parallel copy is compared to the conventional reverse complementary copy. It is found that the parallel copy is more efficient at higher temperatures (up to 90 degrees C). A model for the ribopolymerase, based on the core of the large subunit (LSU) of the ribosome, is described. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have 'poisoned' the ribopolymerase by cross-reacting with the nucleoside-triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes, it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of aminoacylated tRNA-like products. Using base-pairing interactions, some of these molecules might cross-link two ribopolymerases, giving rise to a precursor of the modern ribosome. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors.
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PMID:Transcription and translation in an RNA world. 1700 16

Reverse transcriptase (RT) and integrase (IN) are two essential enzymes that play a critical role in synthesis and integration of the retroviral cDNA, respectively. For human immunodeficiency virus type 1 (HIV-1), RT and IN physically interact and certain mutations and deletions of IN result in viruses defective in early steps of reverse transcription. However, the mechanism by which IN affects reverse transcription is not understood. We used a cell-free reverse transcription assay with different primers and compositions of deoxynucleoside triphosphates to differentially monitor the effect of IN on the initiation and elongation modes of reverse transcription. During the initiation mode, addition of IN stimulated RT-catalyzed reverse transcription by fourfold. The stimulation was specific to IN and could not be detected when the full-length IN was replaced with truncated IN derivatives. The IN-stimulated initiation was also restricted to the template-primer complex formed using tRNA(3)(Lys) or short RNA oligonucleotides as the primer and not those formed using DNA oligonucleotides as the primer. Addition of IN also produced a threefold stimulation during the elongation mode, which was not primer dependent. The stimulation of both initiation and elongation by IN was retained in the presence of an RT trap. Furthermore, IN had no effect on steps at or before template-primer annealing, including packaging of viral genomic RNA and tRNA(3)(Lys). Taken together, our results showed that IN acts at early steps of reverse transcription by increasing the processivity of RT and suppressing the formation of the pause products.
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PMID:Molecular mechanisms by which human immunodeficiency virus type 1 integrase stimulates the early steps of reverse transcription. 1762 89

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.
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PMID:Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. 2044 Aug 79

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qbeta is composed of the viral catalytic beta-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the beta-subunit and the two host proteins to 2.5-A resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the beta-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the beta-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.
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PMID:Structure of the Qbeta replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins. 2053 94

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.
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PMID:Nonnucleoside reverse transcriptase inhibitor-resistant HIV is stimulated by efavirenz during early stages of infection. 2183 88

The majority of the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3' terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3' elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3' region of a plus-strand RNA virus.
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PMID:A local, interactive network of 3' RNA elements supports translation and replication of Turnip crinkle virus. 2234 59


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