E. Dufour et al., Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNA(Lys3), J MOL BIOL, 285(4), 1999, pp. 1339-1346
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) init
iates the synthesis of DNA from the 3' end of its specific primer, tRNA(Lys
3). The regions of tRNA(Lys3) in close contact with RT are well known, whil
e a precise knowledge of the RT regions interacting with tRNA(Lys3) is not
yet available. To address this question we cross-linked the heterodimeric p
66/51 RT to tRNA(Lys3) using cis-aquahydroxydiammine-platinum. Ribonucleopr
otein complexes of molecular masses higher than the p66 subunit were obtain
ed. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucl
eotide (ORN) was mainly found associated to the p66 subunit. This labeled p
66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease.
A highly purified radioactive peptide was obtained after two chromatograph
ic purification steps. Its N-terminal sequence corresponded with amino acid
residues (241)VQPI(244). Using the crystallographic structure of HIV-1 RT,
this peptide was localized at the beta(14)-sheet end, near to the hairpin
formed by beta(12) and beta(13)-sheets ("primer grip") and the alpha(H)-hel
ix. The so called "VQPI peptide" is in the border of the thumb and the palm
subdomains of the p66 subunit. This study palliates the absence of a three
-dimensional structure of the RT-tRNA complex and led to a peptide in inter
action with tRNA(Lys3) present in all HIV-1 RT isolates. (C) 1999 Academic
Press.