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)

The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+. Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E. coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E. coli RNase HI is an excellent substrate binding region because of its sequence and/or location. The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase.
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PMID:Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase. 753 Mar 60

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.
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PMID:HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA. 753 25

The human immunodeficiency virus (HIV) and other retroviruses show extensive genomic variation, which is primarily due to error-prone replication by the viral reverse transcriptase (RT) enzymes. RT errors include misincorporation with subsequent extension of the mismatched terminal base, and extension of realigned primer-template duplexes. Whereas both RT-mediated mechanisms have been extensively studied in vitro, almost no in vivo experiments have been performed. In this work, we analyzed the ability of HIV-1 RT to extend a misaligned tRNA(Lys3) primer in vivo. This tRNA binds with its 3'-terminal 18 nt to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). We constructed a series of mutant viral genomes with small insertions or deletions in the PBS sequence, resulting in misalignment of the tRNA primer. Extension of the misaligned primer did occur with reasonable efficiency for some of the mutants, resulting in reversion to the wild-type viral sequence. The infectivity and reversion frequency of the PBS mutants is therefore a measure of the efficiency of extending a misaligned primer in vivo. Using virion-derived primer-template complexes, we also measured the tRNA-priming efficiency in vitro. The combined results show that HIV-1 RT can elongate a misaligned primer and that the efficiency of primer extension is determined by the extent of the mismatch.
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PMID:Efficient extension of a misaligned tRNA-primer during replication of the HIV-1 retrovirus. 753 60

We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription.
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PMID:Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. 754 Jan 37

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).
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PMID:tRNAs as primer of reverse transcriptases. 754 Dec 50

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at a site in the viral RNA genome which is designated the primer-binding site (PBS). The HIV-1 PBS is an 18-nucleotide sequence that is complementary to the 3'-terminal 18 nucleotides of tRNA(3Lys), which is used as the primer for reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA(3Lys), suggesting that other cellular tRNAs might not function as primers for reverse transcription. To investigate this possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3'-terminal nucleotides of tRNA(1,2Lys), tRNA(Ile), and tRNA(His), which previous studies have identified to be packaged into HIV-1 virions along with tRNA(3Lys). We demonstrate that infectious viruses which utilized tRNA(1,2Lys), tRNA(Ile), and tRNA(His) in reverse transcription can be recovered. However, the appearances of viruses with PBSs complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type PBS complementary to tRNA3(Lys). Furthermore, only the first 9 nucleotides of the 18 nucleotide PBSs were sufficient for HIV-1 to utilize the alternate tRNA primers in reverse transcription, demonstrating that HIV-1 does not require the complete 18-nucleotide PBS to utilize these tRNA primers for reverse transcription. These results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription.
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PMID:Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys). 754 40

The relationship between human immunodeficiency virus (HIV) type 1 reverse transcriptase tG:T mispair formation and base pair stability was investigated using DNA and RNA templates with 15 bp matched or mismatched DNA primers. tG:T mispair formation during primer elongation was undetectable on tDNA-DNA duplexes but occurred with a frequency of 10(-4) on matched tRNA-DNA duplexes. The frequency increased to 7.0 x 10(-4) and 1.3 x 10(-3) on tRNA-DNA duplexes with tG:T mismatches located 6 and 9 bp beyond the polymerization site. From Km values at 37 degrees C, the free energy change upon dissociation (delta G degrees 37) of the tG:T mispair increased from matched to mismatched tRNA-DNA duplexes by 0.36-1.21 kcal/mol. delta G degrees 37 for a correct tG:C pair decreased by 0.06-1.00 kcal/mol. In comparison with DNA-DNA duplexes, thermal melting measurements on RNA-DNA duplexes demonstrated smaller enthalpy (delta delta H degrees = -17.7 to -28.1 kcal/mol) and entropy (delta delta S degrees = -59.3 to -83.4 cal/mol/K) components. A strong entropy-enthalpy compensation resulted in small free energy differences (delta delta G degrees 37 = 0.8 to -2.2 kcal/mol). Thus, although DNA-DNA and RNA-DNA duplexes are of comparable stability in solution, the RNA-DNA duplex presents more facile base pair opening and higher conformational flexibility. The release of helical strain at constant helix stability in RNA-DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G-->A hypermutation.
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PMID:Human immunodeficiency virus type 1 reverse transcriptase tG:T mispair formation on RNA and DNA templates with mismatched primers: a kinetic and thermodynamic study. 755 5

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA molecule that are required for interaction with the RT protein remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified RT to mutant forms of tRNA Lys3. The anticodon loop could be mutated without eliminating RT recognition. However, mutations in the T psi C stem were found to partially interfere with RT binding, and D arm mutants were completely inactive in RT binding. Interestingly, binding of the RT protein to tRNA Lys3 facilitates the subsequent annealing of template strand to the 3'-terminus of the tRNA molecule. Consistent with this finding, we demonstrate that mutant HIV-1 virions lacking the RT protein do contain a viral RNA genome without an associated tRNA Lys3 primer. We also found that a preformed primer tRNA-template complex is efficiently recognized by RT protein in vitro. Extension of the template molecule over the T psi C loop did result in complete inhibition of RT binding, suggesting the presence of additional recognition elements in the T psi C loop. These results, combined with a comparative sequence analysis of tRNA species present in HIV-1 virions and RNA motifs selected in vitro for high affinity RT binding, suggest that RT recognizes the central domain of the tRNA tertiary structure, which is formed by interaction of the D and T psi C loops.
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PMID:Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1. 755 65

T-cell lines (Jurkat and Molt-4) were transduced with retroviral vectors containing a hairpin ribozyme that targets a conserved sequence in the 5' transcribed leader sequence of human immunodeficiency virus (HIV) type 1. Stable cell lines were generated which constitutively and persistently expressed the ribozyme gene driven by either the Moloney retroviral long terminal repeat (LTR) or an internal human tRNA(val) promoter. There was no apparent deleterious effect of long-term ribozyme expression on cell proliferation or viability. Cells expressing ribozyme were resistant to challenge from diverse strains of HIV, including an uncloned clinical isolate. No reverse transcriptase activity or virus infectivity was detectable in the culture supernatants of Jurkat cells expressing the ribozyme driven by the tRNA(val) promoter up to 35 days after challenge with HIV-1/HXB2. Expression of the ribozyme also significantly decreased (by approximately 50- to 100-fold) the efficiency of incoming virus to synthesize viral DNA. These and previously reported results indicate that transfer and expression of the ribozyme gene interfere with both early and late events in the HIV replication cycle and confer long-term resistance to HIV-1 infection.
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PMID:Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1. 758 58

The nucleocapsid protein NCp15 of human immunodeficiency virus type 1 (HIV-1) is a small basic protein with two zinc fingers. It is required for virion morphogenesis and synthesis of proviral DNA. As the first step in our study of the structural domains involved in the various functions of this protein, 18 monoclonal antibodies (MAbs) were isolated. The epitopes of NCp7 recognized by the MAbs were mapped using synthetic peptides representing overlapping sequences and truncated forms of NCp7. These anti-NCp7 MAbs were investigated by ELISA and real-time biospecific interaction analysis (BIAcore). Five classes of anti-NCp7 MAbs were characterized. Three classes (14 MAbs) were directed against continuous epitopes, one in the N-terminal part, another next to the second zinc finger and the third in the C-terminal part of the protein. Two other classes comprised four MAbs reacting only with the entire NCp7 and not with any of the small overlapping peptides used, suggesting that these MAbs were directed against conformational epitopes. The anti-NCp7 MAbs directed against linear epitopes were able to react efficiently with both NCp7 and NCp15, the NCp7 precursor, whereas the anti-NCp7 MAbs directed against conformational epitopes did not react with NCp15. Interestingly, most of the anti-NCp7 MAbs directed against conformational epitopes were capable of inhibiting the tight interaction between NCp7 and the HIV-1 replication primer tRNA(Lys,3). In contrast, most of the MAbs directed against linear epitopes did not inhibit this interaction.
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PMID:Conformational changes between human immunodeficiency virus type 1 nucleocapsid protein NCp7 and its precursor NCp15 as detected by anti-NCp7 monoclonal antibodies. 759 49

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