Structural Basis of RNA Stem-Loop Kissing Interactions

描述（由申请人提供）：核糖核酸（RNA）分子如何折叠成三级结构，蛋白质如何识别特定的RNA结构，以及蛋白质结合如何参与RNA功能，是许多生物过程的中心问题。该提案的广泛目标是阐明形成特定RNA结构折叠（环-环接吻基序）的结构和机制原理，使用多维异源核磁共振（NMR）和荧光光谱作为主要工具。RNA环-环接吻相互作用主要通过环和/或凸起与互补核苷酸序列之间的碱基配对形成，并且是细菌中各种细胞过程、逆转录病毒基因组RNA的二聚化以及某些信使和催化RNA的正确折叠中所涉及的基本结构。了解RNA接吻相互作用如何在分子水平上发挥作用，需要详细研究这些复合物的结构，动力学和蛋白质相互作用。拟议的研究将集中在两个系统上，其中观察到RNA接吻相互作用使RNA二聚化成核，并通过茎链侵入和螺旋交换专门指导进一步的RNA重折叠：HIV-1二聚化起始位点（DIS）基因组二聚体连接和R1的反义调节质粒repA mRNA翻译。辅助蛋白HIV-1核衣壳（NCp 7）和Gag在陪伴DIS接吻二聚体重折叠中的作用也将被研究。具体而言，我们的研究将旨在：1）检查RNA茎环结构在决定repA mRNA-阻遏物RNA接吻相互作用中螺旋交换的方向和程度中的作用; 2）检查RNA接吻二聚体结构和动力学在NCp 7陪伴的HIV-1 DIS成熟中的作用; 3）使用片段标记和荧光/NMR光谱法研究DIS在HIV-1前导序列背景下的二聚化和成熟; 4）分析DIS接吻二聚体的NCp 7和Gag陪伴成熟，并筛选该过程的抑制剂。总体而言，预计这些研究将有助于设计更有效的反义疗法，并为设计抗HIV-1 DIS、NCp 7和/或Gag的抑制剂提供合理的方法，从而促进新的抗病毒疗法的开发。

DESCRIPTION (provided by applicant): How ribonucleic acid (RNA) molecules fold into tertiary structures, how proteins recognize specific RNA structures, and how protein binding is involved in RNA functions, are central questions to a number of biological processes. The broad goal of this proposal is to elucidate structural and mechanistic principles that underlie the formation of a particular RNA structural fold, the loop-loop kissing motif, using multi-dimensional heteronuclear nuclear magnetic resonance (NMR) and fluorescence spectroscopy as the primary tools. RNA loop-loop kissing interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are essential structures involved in a variety of cellular processes in bacteria, in the dimerization of retroviral genomic RNA and in the proper folding of certain messenger and catalytic RNAs. Understanding how RNA kissing interactions function at the molecular level requires detailed studies of the structure, dynamics and protein interactions of these complexes. The proposed studies will focus on two systems in which RNA kissing interactions are observed to nucleate RNA dimerization and specifically direct further RNA refolding through stem strand invasion and helix exchange: the HIV-1 dimerization initiation site (DIS) genomic dimer linkage and the antisense regulation of R1 plasmid repA mRNA translation. The role of the auxiliary proteins HIV-1 nucleocapsid (NCp7) and Gag in chaperoning DIS kissing dimer refolding will also be examined. Specifically, our research will aim to: 1) Examine the role of RNA stem-loop structure in determining the directionality and extent of helix exchange in the repA mRNA-repressor RNA kissing interaction; 2) Examine the role of RNA kissing dimer structure and dynamics in NCp7 chaperoned HIV-1 DIS maturation; 3) Investigate dimerization and maturation of DIS within the context of HIV-1 leader sequences using segmental labeling and fluorescence/NMR spectroscopy; 4) Analyze NCp7 and Gag chaperoned maturation of the DIS kissing dimer and screen for inhibitors of this process. Overall, it is anticipated that these studies will aid in the design of more effective antisense therapies, as well as provide rational approaches for designing inhibitors against HIV-1 DIS, NCp7 and/or Gag that could facilitate development of new antiviral therapeutics.