Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNA(Lys3)

Citation
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
Citations number
41
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
00222836 → ACNP
Volume
285
Issue
4
Year of publication
1999
Pages
1339 - 1346
Database
ISI
SICI code
0022-2836(19990129)285:4<1339:CLOAHR>2.0.ZU;2-6
Abstract
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.