Structural Basis of RNA Stem-Loop Kissing Interactions

描述(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。这一建议的广泛目标是利用多维异核核磁共振(核磁共振)和荧光光谱作为主要工具，阐明形成特定RNA结构折叠(环-环亲吻基序)的结构和机械原理。RNA环-环亲吻作用主要通过环和/或与互补核苷酸序列的凸起之间的碱基配对形成，是参与细菌的各种细胞过程、逆转录病毒基因组RNA的二聚化以及某些信使和催化RNA的适当折叠的基本结构。要了解RNA亲吻相互作用在分子水平上的作用，需要详细研究这些复合体的结构、动力学和蛋白质相互作用。这些研究将集中在两个系统上，在这两个系统中，观察到RNA亲吻相互作用导致RNA二聚化，并通过干链入侵和螺旋交换特异性地指导进一步的RNA折叠：HIV-1二聚化起始点(DIS)基因组二聚体连接和R1质粒REPA mRNA翻译的反义调控。辅助蛋白HIV-1核衣壳(NCp7)和GAG在伴随DIS亲吻二聚体折叠中的作用也将被研究。具体地说，我们的研究旨在：1)检测RNA茎环结构在决定REPA mRNA-阻遏物RNA亲吻相互作用中螺旋交换的方向性和程度中的作用；2)检测RNA亲吻二聚体结构和动力学在NCp7伴随的HIV-1 DIS成熟中的作用；3)使用分段标记和荧光/核磁共振光谱研究DIS在HIV-1前导序列中的二聚化和成熟；4)分析NCp7和Gag结合的DIS亲吻二聚体的成熟并筛选这一过程的抑制剂。总体而言，预计这些研究将有助于设计更有效的反义疗法，并为设计针对HIV-1 DIS、NCp7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.