Mihailescu Mihaela-Rita, Marino John P
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and National Institute for Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1189-94. doi: 10.1073/pnas.0307966100. Epub 2004 Jan 20.
In HIV type 1 (HIV-1), the dimerization initiation site (DIS) is the sequence primarily responsible for initiating the noncovalent linkage of two homologous strands of genomic RNA during viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence and forms a symmetric homodimer through a loop-loop kissing interaction. In a structural rearrangement catalyzed by the HIV-1 nucleocapsid protein (NCp7) and suggested to be associated with maturation of the budded viral particle, the DIS converts from a metastable kissing dimer to an extended duplex. Here, we demonstrate that the DIS kissing dimer displays localized conformational dynamics that result from the specific protonation of the N1 base nitrogen of the DIS loop residue A272 at near-physiological pH. The rate of NCp7-catalyzed maturation of the DIS kissing dimer is also shown to directly correlate with the observed proton-coupled conformational dynamics, where NCp7 is found to convert the dynamic A272 protonated state with a faster rate. Taken together, these results reveal a previously undescribed role for base protonation in modulating local RNA structure and demonstrate a mechanism for promoting the chaperone-mediated structural rearrangement of a kinetically trapped RNA conformational state.
在1型人类免疫缺陷病毒（HIV-1）中，二聚化起始位点（DIS）是病毒组装过程中主要负责启动基因组RNA两条同源链非共价连接的序列。DIS环包含一个自身互补的六核苷酸序列，并通过环-环亲吻相互作用形成对称的同型二聚体。在由HIV-1核衣壳蛋白（NCp7）催化的结构重排中，该重排被认为与出芽病毒颗粒的成熟有关，DIS从亚稳态的亲吻二聚体转变为延伸的双链体。在此，我们证明DIS亲吻二聚体表现出局部构象动力学，这是由DIS环残基A272的N1碱基氮在接近生理pH值下的特定质子化所导致的。DIS亲吻二聚体由NCp7催化的成熟速率也被证明与观察到的质子耦合构象动力学直接相关，其中发现NCp7以更快的速率转换动态的A272质子化状态。综上所述，这些结果揭示了碱基质子化在调节局部RNA结构方面以前未被描述的作用，并证明了一种促进伴侣介导的动力学捕获RNA构象状态结构重排的机制。
