Liang C, Rong L, Götte M, Li X, Quan Y, Kleiman L, Wainberg M A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada.
J Biol Chem. 1998 Aug 14;273(33):21309-15. doi: 10.1074/jbc.273.33.21309.
We have investigated the role of sequences that surround the primer binding site (PBS) in the reverse transcriptase-mediated initiation of (-) strand DNA synthesis in human immunodeficiency virus type 1. In comparisons of reverse transcription initiated from either the cognate primer tRNALys.3 or a DNA primer D-Lys.3, bound to PBS sequences, we observed that a +3 pausing site occurred in both circumstances. However, the initiation reaction with tRNALys.3 was also characterized by a pausing event after incorporation of the first nucleotide. Alteration of sequences at the 5'-end instead of the 3'-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template sequence-dependent. In contrast, the pausing event at the +1 nucleotide position was still present in experiments that employed either of these mutated RNA templates. The mutations at the 5'-end of the PBS also caused a severely diminished rate of initiation and the strong arrest of reactions at the +1 stage when tRNALys.3 was used as primer. Therefore, we propose that the +1 pausing event is an initiation-specific event in regard to reactions primed by tRNALys.3 and that sequences at the 5'-end of the PBS may facilitate the release of reverse transcription from initiation to elongation.
我们研究了人免疫缺陷病毒1型中围绕引物结合位点（PBS）的序列在逆转录酶介导的负链DNA合成起始过程中的作用。在比较从同源引物tRNALys.3或与PBS序列结合的DNA引物D-Lys.3起始的逆转录时，我们观察到在两种情况下都出现了一个+3暂停位点。然而，用tRNALys.3进行的起始反应还具有在掺入第一个核苷酸后出现暂停事件的特征。PBS 5'-端而非3'-端序列的改变导致+3暂停位点消失，这表明该位点依赖于模板序列。相比之下，在使用这些突变RNA模板之一的实验中，+1核苷酸位置的暂停事件仍然存在。当使用tRNALys.3作为引物时，PBS 5'-端的突变还导致起始速率严重降低以及反应在+阶段强烈停滞。因此，我们提出，就由tRNALys.3引发的反应而言，+1暂停事件是一个起始特异性事件，并且PBS 5'-端的序列可能有助于逆转录从起始阶段向延伸阶段的转变。
