STRUCTURAL BASIS OF RNA STEM LOOP KISSING INTERACTIONS

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利用多维异核核磁共振（NMR）光谱和其他生物物理方法阐明在三种不同和独特的系统中形成特定RNA结构褶皱（环环“亲吻”基元）的高分辨率结构和机制原理。RNA环环“亲吻”相互作用主要通过环环和/或具有互补核苷酸序列的凸起之间的碱基配对形成，是参与细菌多种细胞过程反义调节的关键中间结构。这些相互作用还涉及某些mRNA， rRNA和催化RNA结构的适当折叠。作为基因组逆转录病毒RNA二聚化的重要元素，环环“接吻”结构也是抗病毒药物的可行靶点。本研究的具体目的是：1)确定两种新型RNA环环“亲吻”复合物特异性相互作用的结构基础：一种是由HIV-1基因组RNA中二聚化起始位点（DIS）衍生的RNA茎环形成的同二聚体环环“亲吻”复合物，另一种是由参与R1质粒复制控制的mRNA CopA和反意义CopT阻遏转录物衍生的RNA茎环形成的环环“亲吻”复合物；2)确定蛋白质Rom对ColE1复制控制系统衍生的环环“接吻”复合物特异性识别的结构基础。核磁共振光谱结构和动力学研究将辅以停止流动动力学、热熔和凝胶电泳实验，旨在分析野生型和突变型环环“接吻”复合物的热力学稳定性和结合/解离动力学。新的核磁共振方法，特别适用于13C， 15N和/或2H标记的RNA和RNA-蛋白质复合物的大小（10 kDa - 26 kDa），也将被设计，将提供比现有技术更高的精度和灵敏度，并将获得目前RNA分子无法获得的全局远程结构限制。

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 the high-resolution structures and mechanistic principles that underlie the formation of a particular RNA structural fold (the loop-loop "kissing" motif) in three different and unique systems using multi- dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy and other biophysical methods. RNA loop-loop "kissing" interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are the critical intermediate structures involved in the antisense regulation of a variety of cellular processes in bacteria. These interactions are also involved in the proper folding of certain mRNA, rRNA and catalytic RNA structures. As an important element in dimerization of genomic retroviral RNA, the loop-loop "kissing" structure is also a viable target for anti-viral drugs. The specific aims of this research are: 1) to determine the structural basis for specific interaction in two novel RNA loop-loop "kissing" complexes: a homodimeric loop-loop "kissing" complex formed by an RNA stem-loop derived from the dimerization initiation site (DIS) in HIV-1 genomic RNA and a loop-loop "kissing" complex formed by RNA stem-loops derived from the mRNA CopA and antisense CopT repressor transcripts involved in R1 plasmid replication control; and 2) to determine the structural basis for the specific recognition of a loop- loop "kissing" complex derived from the ColE1 replication control system by the protein Rom. NMR spectroscopic structural and dynamical studies will be complemented by stop-flow kinetic, thermal melting and gel electrophoresis experiments designed to assay thermodynamic stability and association/dissociation kinetics of wild type and mutant loop-loop "kissing" complexes. New NMR methods, specifically applicable to 13C, 15N and/or 2H labeled RNA and RNA-protein complexes of sizes (10 kDa - 26 kDa), will also be designed which will provide higher precision and sensitivity than available with existing technology and which will access global long-range structural restraints currently unavailable for RNA molecules.