Arion D, Harada R, Li X, Wainberg M A, Parniak M A
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec, Canada.
Biochem Biophys Res Commun. 1996 Aug 23;225(3):839-43. doi: 10.1006/bbrc.1996.1260.
The transcription initiation primer for HIV-1 is a specific cellular tRNA species, tRNA(Lys3). We used several methods to assess the binding of tRNA by recombinant HIV-1 p51/p66 reverse transcriptase (RT), gel retardation analysis, intrinsic RT protein fluorescence quenching, and nitrocellulose filter binding assays. The binding of tRNA to RT was saturable, implying a distinct site or sites on the enzyme for tRNA interaction. However, this binding was non-selective, with all tRNA isoacceptors and total unfractionated tRNA binding with similar affinity as primer tRNA(Lys3). In contrast, no significant binding of tRNA by RT was noted. Our results show that HIV-1 RT has no specificity for the binding of primer tRNA(Lys3), and imply that factors other than RT sequences may be important for the selective incorporation of primer tRNA into the virion particle.
HIV-1的转录起始引物是一种特定的细胞tRNA种类，即tRNA(Lys3)。我们使用了几种方法来评估重组HIV-1 p51/p66逆转录酶（RT）与tRNA的结合，包括凝胶阻滞分析、RT蛋白固有荧光猝灭以及硝酸纤维素滤膜结合测定。tRNA与RT的结合是可饱和的，这意味着该酶上存在一个或多个与tRNA相互作用的独特位点。然而，这种结合是非选择性的，所有tRNA同工受体和未分级的总tRNA与引物tRNA(Lys3)的结合亲和力相似。相比之下，未观察到RT与tRNA有明显的结合。我们的结果表明，HIV-1 RT对引物tRNA(Lys3)的结合没有特异性，这意味着除RT序列外的其他因素可能对引物tRNA选择性掺入病毒粒子很重要。
