通过单分子 FRET 显微镜研究引物激活信号在 tRNA 退火到 HIV-1 基因组中的作用。
Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy.
机构信息
1Department of Molecular Biology, Aarhus University, Aarhus, Denmark.
出版信息
RNA. 2013 Apr;19(4):517-26. doi: 10.1261/rna.035733.112. Epub 2013 Feb 12.
Abstract
HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.
摘要
HIV-1 逆转录由细胞 tRNAlys3 分子引发，该分子与基因组 RNA 中的引物结合位点 (PBS) 结合。tRNA 分子与引物激活信号 (PAS) 之间的额外相互作用被认为可以调节逆转录的起始。使用荧光供体和受体分子共价连接到模拟 PBS 区域的 RNA 模板的集合和单分子Förster 共振能量转移 (FRET) 测定法检查了 tRNA 退火到 HIV-1 基因组的机制。研究了病毒核衣壳 (NC) 蛋白在 tRNA 退火中的作用。发现热退火和 NC 介导的 tRNAlys3 退火都改变了 FRET 效率，从而改变了 HIV-1 RNA 模板的构象。结果与涉及 tRNAlys3 分子与 HIV-1 基因组中 PAS 序列相互作用的 tRNA 退火模型一致。NC 蛋白可能会刺激 tRNA 分子与 PAS 的相互作用，从而调节逆转录的起始。
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