Publications
1999
Auxilien S., Keith G., Grice S. F. Le, Darlix J. L.
Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription Journal Article
In: J Biol Chem, vol. 274, no. 7, pp. 4412-20, 1999, (0021-9258 Journal Article).
Abstract | BibTeX | Tags: *RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism
@article{,
title = {Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription},
author = { S. Auxilien and G. Keith and S. F. Le Grice and J. L. Darlix},
year = {1999},
date = {1999-01-01},
journal = {J Biol Chem},
volume = {274},
number = {7},
pages = {4412-20},
abstract = {During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.},
note = {0021-9258
Journal Article},
keywords = {*RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism},
pubstate = {published},
tppubtype = {article}
}
1998
Motorin Y., Keith G., Simon C., Foiret D., Simos G., Hurt E., Grosjean H.
The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity Journal Article
In: RNA, vol. 4, no. 7, pp. 856-69, 1998, (1355-8382 Journal Article).
Abstract | BibTeX | Tags: *RNA, cerevisiae, Cloning, Fractions/metabolism, Fungal, Fungal/metabolism, Gov't, Hydro-Lyases/biosynthesis/genetics/*metabolism, Molecular, Mutation, Non-U.S., Plant/metabolism, post-transcriptional, Precursors/*metabolism, Processing, Proteins/biosynthesis, Proteins/biosynthesis/genetics/metabolism, Pseudouridine/*biosynthesis, Recombinant, RNA, Saccharomyces, Specificity, Subcellular, Substrate, Support, Transfer/*metabolism
@article{,
title = {The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity},
author = { Y. Motorin and G. Keith and C. Simon and D. Foiret and G. Simos and E. Hurt and H. Grosjean},
year = {1998},
date = {1998-01-01},
journal = {RNA},
volume = {4},
number = {7},
pages = {856-69},
abstract = {We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.},
note = {1355-8382
Journal Article},
keywords = {*RNA, cerevisiae, Cloning, Fractions/metabolism, Fungal, Fungal/metabolism, Gov't, Hydro-Lyases/biosynthesis/genetics/*metabolism, Molecular, Mutation, Non-U.S., Plant/metabolism, post-transcriptional, Precursors/*metabolism, Processing, Proteins/biosynthesis, Proteins/biosynthesis/genetics/metabolism, Pseudouridine/*biosynthesis, Recombinant, RNA, Saccharomyces, Specificity, Subcellular, Substrate, Support, Transfer/*metabolism},
pubstate = {published},
tppubtype = {article}
}