A model of PSI dimerization: Destabilization of the C-278-G(303) stem-loop by the nucleocapsid protein (NCp10) of MoMuLV
A model of PSI dimerization: Destabilization of the C-278-G(303) stem-loop by the nucleocapsid protein (NCp10) of MoMuLV
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DOI：
10.1021/bi952454s
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发表时间：
1996-07-02
期刊：
影响因子：
2.9
通讯作者：
Paoletti, J
中科院分区：
文献类型：
--
作者：
Girard, PM;deRocquigny, H;Paoletti, J
我们已经证明，在低离子强度（即 100 mM 氯化钠）下，MoMuLV RNA 基因组的跨核苷酸 C-283 至 G(298) 的短自互补序列参与体外 PSI 二聚化过程 [Girard, P.-M., Bonnet-Mathoniere, B., Muriaux, D., & Paoletti, J. (1995) Biochemistry 34, 9785-9794]。为了确定 PSI 结构对 RNA 二聚化的其他贡献，我们研究了二聚化动力学作为包含或不包含自互补序列 C-283-G(298) 的短 RNA 转录本的盐浓度的函数。我们提出，除了该序列在 RNA 二聚化中的关键作用之外，PSI 的 364-565 结构域还可以在体外干扰二聚体形成的起始。涉及 364-565 结构域的分子间环-环识别可以以盐浓度依赖性方式稳定围绕环-环 U-288-A(293) 相互作用构建的瞬时 RNA 二聚体。该二聚体向更稳定的结构演化，主要对应于两个 C-283-G(298) 序列的退火。我们还表明，化学合成的 NCp10 不会修改这些步骤，而是帮助系统跨越与过渡到包含茎环 C-278-G(303) 的稳定 RNA 结构相关的能量障碍。在 NCp10 存在的情况下获得的数据表明，在 37°C 下每个蛋白质的结合位点大小为 9 +/- 1 个核苷酸，二聚体形成的速率常数（即 k(1) = 24 000 +/- 7000 M(-1) s(-1)）增加了 10-20 倍。
We have shown that at low ionic strength (i.e., 100 mM NaCl) a short autocomplementary sequence spanning nucleotides C-283 to G(298) Of MoMuLV RNA genome is involved in the process of PSI dimerization in vitro [Girard, P.-M., Bonnet-Mathoniere, B., Muriaux, D., & Paoletti, J. (1995) Biochemistry 34, 9785-9794]. In order to identify other contributions of the PSI structure to RNA dimerization, we studied the kinetics of dimerization as a function of salt concentration of short RNA transcripts comprising or not the autocomplementary sequence C-283-G(298) We propose that, apart from the crucial role of this sequence in RNA dimerization, the 364-565 domain of PSI can interfere, in vitro, with the initiation of dimer formation. Intermolecular loop-loop recognitions involving the 364-565 domain could stabilize, in a salt concentration-dependent manner, a transient RNA dimer built around the loop-loop U-288-A(293) interaction. This dimer evolves toward a more stable structure which mainly corresponds to the annealing of two C-283-G(298) sequences. We also show that chemically synthesized NCp10 does not modify these steps but rather helps the system to pass over the energy barriers associated with the transition to stable RNA structures comprising the stem-loop C-278-G(303). Data obtained in the presence of NCp10 suggest a binding site size of 9 +/- 1 nucleotides per protein at 37 degrees C and a 10-20-fold increase in the rate constant (i.e., k(1) = 24 000 +/- 7000 M(-1) s(-1)) of dimer formation.