Lanchy J M, Isel C, Keith G, Le Grice S F, Ehresmann C, Ehresmann B, Marquet R
UPR 9002 du CNRS, IBMC, 67084 Strasbourg cedex, France.
J Biol Chem. 2000 Apr 21;275(16):12306-12. doi: 10.1074/jbc.275.16.12306.
Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.
人类免疫缺陷病毒1型（HIV-1）逆转录的起始需要形成一个包含病毒RNA（vRNA）、tRNA3(Lys)和逆转录酶（RT）的复合物。vRNA和引物tRNA3(Lys)除了引物3'端与引物结合位点（PBS）退火外，还形成了几种分子间相互作用。这些相互作用对于逆转录起始的效率和特异性至关重要。然而，由于它们位于PBS的上游，随着DNA合成的进行，它们必须解开。在这里，通过酶促探测跟踪了逆转录起始过程中复合物的动力学。我们的数据揭示了vRNA.tRNA3(Lys)复合物的三级结构与逆转录酶（RT）在距离聚合位点一定距离处的相互作用。起始复合物的结构允许RT与聚合位点上游多达20个核苷酸的模板链相互作用。相反，RT添加核苷酸改变了复合物在距离催化位点10-14个核苷酸处的三级结构。引物延伸后，病毒序列从tRNA解离时，在复合物表面暴露。然而，对应的tRNA序列则被埋入复合物内部。令人惊讶的是，当突变阻止初始复合物中的分子间相互作用时，它们会暴露出来，这表明tRNA的命运取决于初始复合物的三级结构。
