UNIPROT:Q96FX7 - FACTA Search

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Query: UNIPROT:Q96FX7 (tRNA)
26,753 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3'-OH group of the 3'-terminal adenosine residue of tRNA(Lys-3). In this report, we synthesized tRNA(Lys-3) of which the 3'-terminal adenosine residue lacks either a 2'-OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cell-free system consisting of a 141-base RNA template and purified HIV-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'-deoxyadenosine was able to initiate the cDNA synthesis, while the tRNA with the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3'-OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2'-OH group of an internal guanosine residue of a template RNA.
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PMID:Specificity of priming reaction of HIV-1 reverse transcriptase, 2'-OH or 3'-OH. 750 35

The fluorescent nucleotide analog, 2',3'-trinitrophenyladenosine-5'-triphosphate (TNP-ATP), was utilized to quantify the affinities of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) for its substrates. Interaction of this probe with the enzyme brings about a twofold increase in the magnitude of fluorescence emission from the probe, and a blue-shift in wavelength maximum, from 561 to 553 nm. TNP-ATP binds HIV-1 RT with a dissociation constant of 21 microM. The presence of millimolar levels of deoxynucleoside triphosphates or micromolar levels of an oligonucleotide primer analogue, p(dT)12-18, suppressed this enhancement of fluorescence. The fact that inhibition was achieved with much lower levels of primer than of dNTPs suggests that TNP-ATP is a probe for the binding site of primer on the enzyme, rather than that of deoxynucleoside triphosphate. In support of this, the effect of TNP-ATP on the kinetics of DNA synthesis catalyzed by the enzyme indicated that the probe is a competitive inhibitor with respect to template-primer. The ability of primers and primer analogs to reverse the fluorescence enhancement was determined, and the corresponding affinities of these compounds for reverse transcriptase were calculated. The affinity increased with primer length, increasing more than 50-fold from a span of 5 to 15 nucleotide residues. The interaction of polydeoxynucleotides was consistent with a model in which the enzyme bound at adjacent internal sites of about 15 residues in length. Several mammalian and bacterial transfer RNA primers were tested, including the natural primer, tRNA(3Lys). The affinities were found to be between 0.55 and 1.2 microM, with no obvious selectivity for the natural primer, which had a Kd of 0.79 microM. These results are discussed within the context of data for HIV-1 RT obtained by other methodologies.
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PMID:Studies on primer binding of HIV-1 reverse transcriptase using a fluorescent probe. 750 89

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site. 750 99

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.
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PMID:Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H. 750 4

Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides.
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PMID:Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis. 751 65

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA(3Lys) the primer tRNA in HIV-1, and members of the tRNA(1,2Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively incorporated into HIV-1 particles. We have measured the ratio of tRNA(3Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3Lys) per two copies of genomic RNA. We have also obtained evidence that the Pr160gag-pol precursor is involved in primer tRNA(3Lys) incorporation into virus. First, selective tRNA(Lys) incorporation and wild-type amounts of tRNA(3Lys) were maintained in a protease-negative virus unable to process Pr55gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed Pr55gag protein did not selectively incorporate tRNA(Lys), while virions containing both unprocessed Pr55gag and Pr160gag-pol proteins demonstrated select tRNA(3Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase sequences and approximately one-third of the integrase sequence in the Pr160gag-pol precursor resulted in the loss of selective tRNA incorporation and an eightfold decrease in the amount of tRNA(3Lys) per two copies of genomic RNA. We have also confirmed herein finding of a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNA(Lys).
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PMID:Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. 751 Nov 67

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.
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PMID:Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. 751 78

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.
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PMID:Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. 751 21

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).
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PMID:Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication. 752 16

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
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PMID:Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization. 752 8

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