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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutants of avian sarcoma virus which lack a functional DNA polymerase were found to be nonselective in the incorporation of host cell tRNA's into virus particles. In contrast, mutants which possess a functional DNA polymerase but lack the viral genome RNA contained a specific subset of the host cell tRNA population, indistinguishable from that of the wild-type virus. Thus the reverse transcriptase, and not the viral RNA, is probably the major factor determining which tRNA's are incorporated into avian sarcoma virus particles. Supporting evidence was obtained in an in vitro binding assay between purified reverse transcriptase and unfractionated cellular tRNA's. However, the subset of tRNA's which associated with the genome in the 70S complex was determined primarily by the viral RNA. In the absence of DNA polymerase, the 70S RNA complex in mature virus particles contained the normal complement of associated tRNA's with the exception of tRNATrp, the primer for RNA-directed DNA synthesis.
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PMID:Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles. 616 35

The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.
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PMID:Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I). 616 82

The role of reverse transcriptase in selective encapsidation of the murine leukemia virus (MuLV) tRNA primer, tRNAPro, was investigated by examining the tRNA composition of several nonconditional pol mutants. One mutant, clone 23, which contains an altered polymerase about 40% smaller than the wild-type enzyme (B. I. Gerwin et al., J. Virol. 31:741-751, 1979) had a typical viral tRNA pattern, including normal levels of tRNAPro in free and 70S-associated 4S RNA. Another class of mutants, produced by Moloney murine leukemia virus-infected cell clone M13 and subclone M13/1, does not contain any detectable polymerase protein (A. Shields et al., Cell 14:601-609, 1978) and was found to have reduced amounts of tRNAPro in free 4S RNA. However, the level of tRNAPro associated with the genome was normal in the mutant virions. These results suggest that the reverse transcriptase protein is involved in the initial selection of tRNA primer during virus assembly, but not in the subsequent association of this tRNA with genomic RNA.
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PMID:Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. 616 33

Tryptophanyl-tRNA can be used as a primer for RNA-dependent DNA synthesis by avian reverse transcriptase. We have determined that whereas the retroviral polymerase is not by itself capable of deacylating Trp-tRNATrp, the ester linkage between the 3' OH of the ribose moiety and the aminoacid can be very efficiently hydrolyzed when both the polymerase and the viral 35 S RNA are present.
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PMID:Avian myeloblastosis reverse transcriptase deacylates tryptophanyl-tRNA. 618 23

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed. The extensive modification of tRNATrp with acrylonitrile led to a marked loss of priming activity by tRNATrp if the annealing between primer and template was performed at 37 degrees C, while the annealing of cyanoethylated tRNA with the viral genome at 75 degrees C gave almost normal levels of cDNA synthesis. We have also studied the priming behaviour of tRNATrp, modified by incorporation of various analogs of adenosine. Only tRNATrp-2'dA was active in cDNA initiation; 3'dA, 3'NH2-3'dA, and primer tRNA with formycin in the 3' end showed low or nonexistent priming activity.
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PMID:Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions. 618 58

The basis of the specific binding of tRNATrp by avian myeloblastosis virus reverse transcriptase was studied by chemical and enzymatic modification of the RNA. Binding does not depend on recognition of the tryptophan anticodon since molecules cleaved in the anticodon are stably bound by the enzyme. Modification of pseudouridine residues in the tRNA destroys binding to reverse transcriptase. These results are consistent with a model in which reverse transcriptase-tRNATrp interaction occurs not at the anticodon, but at regions in the tRNA which contain or are stabilized by pseudouridine residues.
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PMID:Structural features required for the binding of tRNATrp to avian myeloblastosis virus reverse transcriptase. 619 93

The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.
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PMID:Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases. 620 Aug 30

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.
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PMID:Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus. 632 19

Probably the only RNA molecule to be imported into yeast mitochondria is tRNA1Lys. The tRNA is not aminoacylated in mitochondria and its function remains unclear. I have noted that after partial unwinding the tRNA1Lys (more of less in the position, where reverse transcriptase unwinds primer tRNAs) it is possible to fit the donor regions of seven different mitochondrial mosaic genes introns to the unwound part of tRNA. The exact splice site is in all cases found according to the following rule: base pair, cross, locate first m2G X U, cut, base pair. Possible exception is the 5th intron of cob-box where the boundary indicated is not the one accepted in literature, nevertheless the boundary suggested here seems possible. The complementarity suggested permits accurate prediction of 4 (5 if b15 counts) "uncanonical" donor boundaries. The four remaining introns are fitted by the author into looped structures held by unwound tRNA1Lys. The two groups of introns suggested coincide exactly with the Dujonian classes I and II, his classification being based on inner clusters of homology rather than intron/exon boundaries used here. A quite impressive fit was found also between tRNA1Lys and box 9, a sequence needed for splicing of a 1st class intron b14 RNA. The sequences playing probably a crucial role in splicing of 2nd class introns: GGGGG and GGCUC are both complementary to a 3'-CCCCC--GAG-5'-sequence in the GT psi C loop+arm of tRNA1Lys. Used or not in splicing tRNA1Lys curiously enough looks like a compendium of nucleotide sequences needed to handle the mitochondrial mosaic genes RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Possible role of tRNA1Lys in the splicing of mitochondrial transcripts in yeasts]. 655 17

Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.
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PMID:Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains. 748 84


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