NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription.
Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry psis, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.
Tisné
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Laboratoire de Cristallographie et Résonance Magnétique Nucléaire Biologiques, Faculté de Pharmacie, Centre National de la Recherche Scientifique Equipe Postulante 2075, Paris, France. tisne@pharmacie.univ-paris5.fr
Rigourd
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M
Marquet
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R
Ehresmann
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Dardel
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F
D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007455:Iodine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014779:Virus Replication
D004279:DNA, Viral; C034477:RNA primers; D012357:RNA, Transfer, Lys; C039919:RNA, recombinant; D012313:RNA; D007455:Iodine