Laboratorie de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401, Illkirch, Cedex, France.
Nucleic Acids Res. 2010 Jun;38(11):3632-42. doi: 10.1093/nar/gkq094. Epub 2010 Feb 18.
The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.
多功能 HCV 核心蛋白由一个亲水的 RNA 相互作用的 D1 结构域和一个与膜和脂滴相互作用的疏水的 D2 结构域组成。已经发现核心的 D1 结构域具有核酸退火和链转移的性质。为了进一步了解这些伴侣性质，我们研究了 D1 结构域和包含 D1 碱性簇的两个肽如何协助互补的规范核酸的退火，这些核酸对应于 HIV-1 反式激活反应元件 TAR 及其互补 cTAR 的 DNA 序列。发现核心肽至少增强了三个数量级的 cTAR-dTAR 退火动力学。退火速率不受 dTAR 环的修饰影响，但强烈受到 cTAR 茎末端稳定的影响，这表明核心定向的退火反应是通过 cTAR 和 dTAR 的末端碱基启动的。鉴定了两种动力学途径，存在一个快速的预平衡中间体，然后缓慢转化为最终的延伸双链体。快速和慢速途径的区别在于碱基对的数量，这些碱基对应该被融化以引发中间体。这三个肽的作用相似，证实了核心伴侣性质主要由其碱性簇支持。
