NMR and biochemical characterization of recombinant human tRNA₃^Lys expressed in Escherichia coli: Identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription

Abstract

Reverse transcription of HIV-1 viral RNA uses human tRNA₃^Lys 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 tRNA₃^Lys 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 tRNA₃^Lys 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.