Turner Kevin B, Brinson Robert G, Yi-Brunozzi Hye Young, Rausch Jason W, Miller Jennifer T, Le Grice Stuart F J, Marino John P, Fabris Daniele
University of Maryland Baltimore County, Baltimore, MD, USA.
Nucleic Acids Res. 2008 May;36(8):2799-810. doi: 10.1093/nar/gkn129. Epub 2008 Apr 9.
The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.
典型核酸配体与HIV-1多聚嘌呤序列（PPT）RNA:DNA杂交体以及类似的DNA:DNA、RNA:RNA和交换杂交底物之间的相互作用，被用于探究PPT的结构特征，这些特征有助于其被逆转录酶（RT）特异性识别和加工。嵌入性和沟结合配体的结果表明，野生型PPT杂交体不包含任何可能有助于其与RT相互作用特异性的显著独特沟几何形状和/或堆积排列。相比之下，新霉素优先且选择性地结合到靠近5'(rA)(4):(dT)(4)序列和3' PPT-U3连接处的PPT上。HIV-1 RT与PPT复合物的核磁共振数据表明，RT与新霉素在PPT上突出显示的相同区域内接触。这些观察结果，以及这些位点间距合适以允许与核糖核酸酶H（RNase H）活性位点和p66 RT亚基拇指结构域中的残基相互作用这一事实，表明尽管RT用于与核酸底物接触的裂隙很长，但这些位点提供了离散的结合单元，协同作用不仅决定了PPT的特异性识别，还决定了其在杂交结构上的方向。
