Lanchy J M, Keith G, Le Grice S F, Ehresmann B, Ehresmann C, Marquet R
Unité Propre de Recherche 9002, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France.
J Biol Chem. 1998 Sep 18;273(38):24425-32. doi: 10.1074/jbc.273.38.24425.
HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA3Lys, and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primer-template complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA3Lys. The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA3Lys. The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix alphaH in the p66 thumb subdomain and the RNase H domain of RT, respectively.
HIV-1逆转录酶（RT）利用RNA寡聚物引发DNA合成。逆转录的起始需要HIV-1 RNA、引物tRNA3Lys和RT之间的特定相互作用。我们之前已经表明，寡脱氧核糖核苷酸的延伸（一种模拟延伸的情况）是非特异性的，并且在RT从引物-模板复合物的聚合速率和解离速率方面与起始不同。在这里，我们使用复制中间体来分析从起始阶段到延伸阶段的转变。我们发现，tRNA 3'端的2'-羟基对聚合和解离速率常数的影响有限。相反，在向tRNA3Lys添加第六和第七个核苷酸之间，聚合速率增加了3400倍。当使用寡核糖核苷酸而非寡脱氧核糖核苷酸作为引物时，观察到了相同的聚合速率增加。同时，在向tRNA3Lys添加第17和第19个核苷酸之间，RT从引物-模板复合物的解离速率降低了30倍。聚合和解离速率很可能分别由引物链与p66拇指亚结构域中的αH螺旋以及RT的RNase H结构域的相互作用所控制。
