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，是许多生物过程的核心问题。本提案的主要目标是阐明形成特定RNA结构褶皱的结构和机制原理，即环环亲吻基元，使用多维异核核磁共振（NMR）和荧光光谱作为主要工具。RNA环环亲和作用主要通过环和/或凸起与互补核苷酸序列之间的碱基配对形成，是细菌中多种细胞过程、逆转录病毒基因组RNA二聚化以及某些信使RNA和催化RNA适当折叠中必不可少的结构。要了解RNA亲吻相互作用在分子水平上是如何起作用的，需要对这些复合物的结构、动力学和蛋白质相互作用进行详细的研究。这些研究将集中在两个系统上，其中RNA亲吻相互作用被观察到形成RNA二聚化，并通过茎链入侵和螺旋交换特异性地指导进一步的RNA重折叠：HIV-1二聚化起始位点（DIS）基因组二聚体连锁和R1质粒repA mRNA翻译的反义调控。辅助蛋白HIV-1核衣壳（NCp7）和Gag在陪伴DIS亲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)研究RNA茎环结构在决定repA mrna -抑制RNA接吻相互作用中螺旋交换的方向性和程度中的作用；2)研究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)利用片段标记和荧光/核磁共振光谱研究HIV-1先导序列中DIS的二聚化和成熟；4)分析NCp7和Gag对DIS亲和二聚体成熟的影响，筛选该过程的抑制因子。总之，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.