Reverse transcription of HIV-1 viral RNA uses human
tRNA3Lys 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 tRNA3Lys 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 tRNA3Lys
subspecies, and their modified nucleotide contents have been
characterized by a combination of NMR and biochemical techniques.
Both species carry Ψs, 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,
Ψ27, 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.