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1996
Lanchy J M, Ehresmann C, Grice S F Le, Ehresmann B, Marquet R
Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription Journal Article
In: EMBO J, vol. 15, no. 24, pp. 7178-7187, 1996, ISBN: 9003793, (0261-4189 Journal Article).
Abstract | Links | BibTeX | Tags: DNA Primers HIV-1/*enzymology HIV-1 Reverse Transcriptase/*metabolism Kinetics Protein Binding Support, Genetic, Genetic *Transcription, MARQUET, Non-U.S. Gov't Support, P.H.S. Templates, U.S. Gov't, Unité ARN
@article{,
title = {Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription},
author = {J M Lanchy and C Ehresmann and S F Le Grice and B Ehresmann and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9003793},
isbn = {9003793},
year = {1996},
date = {1996-01-01},
journal = {EMBO J},
volume = {15},
number = {24},
pages = {7178-7187},
abstract = {We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.},
note = {0261-4189
Journal Article},
keywords = {DNA Primers HIV-1/*enzymology HIV-1 Reverse Transcriptase/*metabolism Kinetics Protein Binding Support, Genetic, Genetic *Transcription, MARQUET, Non-U.S. Gov't Support, P.H.S. Templates, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.
1993
Isel C, Marquet R, Keith G, Ehresmann C, Ehresmann B
Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription Journal Article
In: J Biol Chem, vol. 268, no. 34, pp. 25269-25272, 1993, ISBN: 7503978, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: Anticodon/genetics/metabolism Base Sequence Binding Sites Comparative Study DNA Primers HIV-1/genetics/*metabolism HIV-1 Reverse Transcriptase HIV-2/genetics/metabolism Molecular Sequence Data Nucleic Acid Conformation RNA, Genetic, Genetic *Transcription, Lys/*metabolism RNA, MARQUET, Non-U.S. Gov't Templates, Transfer, Unité ARN, Viral/*biosynthesis RNA-Directed DNA Polymerase/*metabolism SIV/genetics/metabolism Support
@article{,
title = {Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription},
author = {C Isel and R Marquet and G Keith and C Ehresmann and B Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7503978},
isbn = {7503978},
year = {1993},
date = {1993-01-01},
journal = {J Biol Chem},
volume = {268},
number = {34},
pages = {25269-25272},
abstract = {In all retroviruses, reverse transcription is primed by a tRNA whose 3' end 18 nucleotides are complementary to the so called viral primer binding site. Previous work showed that reverse transcription of HIV-1 RNA is initiated by tRNA(3Lys). Using a variety of chemical and enzymatic structural probes, we investigated the interactions between HIV-1 RNA and its natural primer tRNA(3Lys). In addition to the predictable contacts between the viral primer binding site and the 3' end of tRNA(3Lys), a specific interaction takes place between an A-rich loop located upstream of the primer binding site region and the anticodon loop of tRNA(3Lys). This AAAA/Umcm5s2UUU loop-loop interaction is not observed when the natural primer is replaced by an in vitro synthesized tRNA(3Lys) transcript. Furthermore, dethiolation of the modified nucleotide mcm5s2U at position 34 of tRNA(3Lys) strongly destabilizes this interaction. Sequence and structure comparisons indicate that the primer/template loop-loop interaction is conserved in all HIV-1 isolates, and possibly also in HIV-2 and SIV.},
note = {0021-9258
Journal Article},
keywords = {Anticodon/genetics/metabolism Base Sequence Binding Sites Comparative Study DNA Primers HIV-1/genetics/*metabolism HIV-1 Reverse Transcriptase HIV-2/genetics/metabolism Molecular Sequence Data Nucleic Acid Conformation RNA, Genetic, Genetic *Transcription, Lys/*metabolism RNA, MARQUET, Non-U.S. Gov't Templates, Transfer, Unité ARN, Viral/*biosynthesis RNA-Directed DNA Polymerase/*metabolism SIV/genetics/metabolism Support},
pubstate = {published},
tppubtype = {article}
}
In all retroviruses, reverse transcription is primed by a tRNA whose 3' end 18 nucleotides are complementary to the so called viral primer binding site. Previous work showed that reverse transcription of HIV-1 RNA is initiated by tRNA(3Lys). Using a variety of chemical and enzymatic structural probes, we investigated the interactions between HIV-1 RNA and its natural primer tRNA(3Lys). In addition to the predictable contacts between the viral primer binding site and the 3' end of tRNA(3Lys), a specific interaction takes place between an A-rich loop located upstream of the primer binding site region and the anticodon loop of tRNA(3Lys). This AAAA/Umcm5s2UUU loop-loop interaction is not observed when the natural primer is replaced by an in vitro synthesized tRNA(3Lys) transcript. Furthermore, dethiolation of the modified nucleotide mcm5s2U at position 34 of tRNA(3Lys) strongly destabilizes this interaction. Sequence and structure comparisons indicate that the primer/template loop-loop interaction is conserved in all HIV-1 isolates, and possibly also in HIV-2 and SIV.
