HIV-1 nucleocapsid protein NCp7 and its RNA stem loop 3 partner: rotational dynamics of spin-labeled RNA stem loop 3.
HIV-1 nucleocapsid protein NCp7 and its RNA stem loop 3 partner: rotational dynamics of spin-labeled RNA stem loop 3.
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DOI：
10.1021/bi800602e
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发表时间：
2008-09
期刊：
影响因子：
2.9
通讯作者：
Xiangmei Xi;Yan Sun;C. Karim;V. Grigoryants;C. Scholes
中科院分区：
文献类型：
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作者：
Xiangmei Xi;Yan Sun;C. Karim;V. Grigoryants;C. Scholes
在纳秒时间范围内探测了 5' 位置自旋标记的 20 聚体 HIV-1 RNA 茎环 3 的翻滚动力学。这种 RNA 与 HIV-1 核衣壳锌指蛋白 1-55 NCp7 相互作用，专门的停流 EPR 揭示了探针固定从毫秒到秒的伴随动力学。 RNA 茎环 3 在 HIV 中高度保守，而 NCp7 对于 HIV-RNA 包装和退火至关重要。 5' 探针不会干扰 RNA 熔解或通过凝胶位移和荧光监测的 NCp7/RNA 相互作用。 5' 标记的 RNA 以亚纳秒各向同性相关时间（室温下约 0.60 ns）翻滚，反映了探针的局部粘度独立键旋转和 40-60% RNA 的粘度依赖性扩散。 NCp7 以 1:1 的比例与自旋标记的 RNA 茎环 3 结合，使自旋标记的翻滚时间增加了约 40%。在低离子强度下，NCp7 与 RNA 的比例 > 或 = 3（即 NCp7 与核苷酸的比例或 = 3:1 复合物也需要完整的 Zn 指）。NCP7/RNA 以 4:1 比例混合的停流 EPR 动力学显示 NCp7 与 RNA 茎环 3 相互作用的主要阶段发生在 4 ms 内，第二阶段发生的时间常数约为 30 ms，固定速度可能较慢，伴随着大型复合物的形成，这项工作在数秒内进行，为自旋标记研究寡核苷酸-蛋白质复合物指明了道路，特别是那些缺乏精确化学计量的复合物，这是病毒包装和基因组制造所必需的。
The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.