Single-Molecule Spectroscopy of HIV-1 Replication Complexes

几个核蛋白复合物的单分子光谱研究表明，HIV-1逆转录酶（RT）假设不同的方向，这取决于核酸底物的性质。一个意想不到的结果是，我们观察到非核苷类RT抑制剂（NNRTI）诱导酶结合的多嘌呤道（PPT）的方向与启动DNA合成不相容。随后的研究检查了tRNA引发的（-）链DNA合成的起始，表明引物结合位点下游的碱基配对U 5-IR茎控制酶的方向，并且这种起始障碍可以通过包括HIV-1核衣壳（NC）蛋白来减轻。将继续进行单分子光谱分析，以检查含NC的Gag前体和RT突变对（a）HIV-1基因组上的tRNA引发起始，（B）tRNALys，相关慢病毒RNA上的3引发起始，（c）在中心终止序列（CTS）处终止的动力学，和（d）γ逆转录病毒RT的构象动力学。这些项目将与新的策略相结合，用于在含有AZ的RT亚基内位点特异性放置荧光团。Phe通过点击化学。

Single-molecule spectroscopy studies of several nucleoprotein complexes demonstrated that HIV-1 reverse transcriptase (RT) assumes different orientations, depending on the nature of the nucleic acid substrate. An unexpected outcome was our observation that nonnucleoside RT inhibitors (NNRTIs) induce enzyme binding to the polypurine tract (PPT) in an orientation incompatible with initiating DNA synthesis. Subsequent studies examined tRNA-primed initiation of (-) strand DNA synthesis, showing that the base-paired U5-IR stem immediately downstream of the primer binding site controls enzyme orientation and that this barrier to initiation can be alleviated by including the HIV-1 nucleocapsid (NC) protein. Single-molecule spectroscopy will be continued to examine the effect of NC-containing Gag precursors and RT mutations on (a) tRNA-primed initiation on the HIV-1 genome, (b) tRNALys,3-primed initiation on related lentiviral RNAs, (c) dynamics of termination at the central termination sequence (CTS), and (d) conformational dynamics of a gammaretroviral RT. These projects will be combined with new strategies for site-specific placement of fluorophores within RT subunits containing AZ-Phe via Click chemistry.