NMR and biochemical characterization of recombinant human tRNA(3)(Lys) 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(3)(Lys) as a prime r. Some of the modified nucleotides carried by this tRNA must play a key ro le in the initiation of this process, because unmodified tRNA produced in v itro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we ha ve designed a recombinant system that allows tRNA(3)(Lys) expression in Esc herichia coli. Because of their high level of overexpression, some modifica tions are incorporated at substoichiometric levels, We have purified the tw o major recombinant tRNA(3)(Lys) subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical te chniques. Both species carry Psis, Ds, T,t(6)A, and m(7)G. Differences are observed at position 34, within the anticodon, One fraction lacks the 5-met hylaminomethyl group, whereas the other lacks the 2-thio group. Although th e s(2)U(34)-containing recombinant tRNA is a less efficient primer, it pres ents most of the characteristics of the mammalian tRNA, On the other hand, the mnm(5)U(34)-containing tRNA has a strongly reduced activity. Our result s demonstrate that the modifications that are absent in E. coli (m(2)G(10), Psi (27), m(5)C(48), m(5)C(49), and m(1)A(58)) as well as the mnm(5) 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.