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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.
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PMID:Initiator methionine tRNA is essential for Ty1 transposition in yeast. 131 82

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.
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PMID:Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. 245 20

Reverse transcription of the yeast retrotransposon Ty1 is primed by the cytoplasmic initiator methionine tRNA (tRNA(iMet)). The primer tRNA(iMet) is packaged inside virus-like particles (VLPs) and binds to a 10 nucleotides minus-strand primer binding site, the (-)PBS, complementary to its 3' acceptor stem. We have found that three short sequences of the Ty1 RNA (box 1, box 2.1 and box 2.2) located 3' to the (-)PBS are complementary to other regions of the primer tRNA(iMet) (T psi C and DHU stems and loops). Reconstitution of reverse transcription in vitro with T7 transcribed Ty1 RNA species and tRNA(iMet) purified from yeast cells shows that the boxes do not affect the efficiency of reverse transcription. Thus the role of the boxes on packaging of the primer tRNA(iMet) into the VLPs was investigated by analysing the level of tRNA(iMet) packaged into mutant VLPs. Specific nucleotide changes in the (-)PBS or in the boxes that do not change the protein coding sequence but disrupt the complementarity with the primer tRNA(iMet) within the VLPs. We propose that base pairing between the primer tRNA(iMet) and the Ty1 RNA is of major importance for tRNA(iMet) packaging into the VLPs. Moreover the intactness of the boxes is essential for retrotransposition as shown by the transposition defect of a Ty1 element harboring an intact (-)PBS and mutated boxes.
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PMID:Yeast Ty1 retrotransposon: the minus-strand primer binding site and a cis-acting domain of the Ty1 RNA are both important for packaging of primer tRNA inside virus-like particles. 752 35

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5' of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5' end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.
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PMID:Cleavage specificities of Moloney murine leukemia virus RNase H implicated in the second strand transfer during reverse transcription. 759 16

Nucleocapsid protein NCp10 of murine leukemia virus (MuLV) is encoded by the 3' domain of gag and contains a zinc finger of the form Cys-X2-Cys-X4-His-X4-Cys flanked by basic amino acids. In the course of virus assembly, NCp10 is necessary for core formation, and the zinc finger flanked by the basic residues is required for the packaging of the genomic RNA dimer. In vitro, NCp10 exhibits strong nucleic acid binding and annealing activities that appear to be critical for virus infectivity since NCp10 promotes dimerization of the viral RNA containing the E/DLS packaging-dimerization signal and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (PBS). Recent in vitro studies have suggested that NCp10 may also play a role in proviral DNA synthesis. To investigate the function of NCp10 in proviral DNA synthesis in vivo, we developed a simple and convenient genetic packaging system consisting of two DNA constructs expressing the packaging components gag-pol and env of Friend MuLV and a Moloney MuLV-based lacZ vector with either the MuLV E+ or a rat VL30 E packaging signal. This system allowed us to examine the consequences of a set of mutations in NCp10 on a single round of recombinant virus replication. Most mutations in the N- or C-terminal domain of NCp10 do not significantly alter infectivity, while those in the zinc finger drastically impair infectivity. Analysis of the viral RNA content in virions showed that all mutations in the zinc finger decrease but do not abolish packaging of the recombinant genome. Interestingly enough, mutation of Y-28 to S (mutation Y28S) in the zinc finger results in RNA packaging at a level similar to that observed upon deletion of three prolines and three arginines in the C-terminal domain of NCp10 (mutant delta PR3). However, mutant Y28S is noninfectious while mutant delta PR3 is only threefold less infectious than the wild-type virus, which prompted us to examine the role of NCp10 protein in proviral DNA synthesis in vivo using these nucleocapsid mutants. PCR amplification was used to analyze viral DNA synthesized in newly infected cells, and results indicate that the Y28S zinc finger mutation impairs reverse transcription, thus suggesting that the nucleocapsid protein zinc finger plays a key role in proviral DNA synthesis in vivo.
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PMID:The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo. 870 95

The interactions between the Reverse Transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and the natural tRNA(Lys3) primer for initiation of viral DNA synthesis were examined. We constructed a set of HIV-1 RNA templates in which the wild-type primer binding site (PBS(Lys3)) is replaced by sequences complementary to tRNA(lle), tRNA(Lys1,2), tRNA(Phe), tRNA(Pro) or tRNA(Trp) and tested the ability of RT enzymes of different retroviral species to initiate cDNA synthesis from self versus non-self tRNA primers. We demonstrate that initiation of HIV-1 reverse transcription is a specific process that is most efficient with the self tRNA(Lys3) primer. Interestingly, the property of HIV-1 RT to discriminate against non-self tRNA primers is lost upon extension of the tRNA by only two deoxyribonucleotides. Furthermore, selective tRNA priming by HIV-1 RT was not observed with viral RNA-tRNA(Lys3) duplexes isolated from HIV-1 virion particles, suggesting that the majority of tRNA(Lys3) primers annealed to viral RNA in particles is extended by a variable number of deoxyribonucleotides. This result indicates that reverse transcription is initiated relatively early in nascently assembled virions.
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PMID:HIV-1 reverse transcriptase discriminates against non-self tRNA primers. 895 74

We constructed a set of mammalian tRNA(Lys)3-pseudogenes, whose pre-tRNAs are processing-deficient in HeLa extract. The 3'-flanking region was designed partially or completely complementary to the PBS-flanking nucleotides of the U5-region of HIV-1 genomic RNA. We show that only the pre-tRNA with completely hybridized 3'-flanking region is efficiently extended by the HIV-1 reverse transcriptase, whereas the partially complementary pre-tRNA(Lys)3, the 3'-terminal 10 nucleotides of which are not hybridized to the viral template, is unable to prime the cDNA-synthesis.
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PMID:Mammalian tRNA(Lys)3 and pre-tRNA(Lys)3 variants as primers and inhibitors of viral cDNA synthesis by HIV reverse transcriptase in vitro. 958 14

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.
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PMID:The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site. 1019 21

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.
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PMID:The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription. 1041 Nov 36

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).
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PMID:Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication. 1063 32


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