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中心问题。 该提议的广泛目标是使用多维异谱核磁共振（NMR）光谱和其他生物物理方法阐明在三种不同且独特的系统中形成特定RNA结构折叠（环-环“接吻”基序）的高分辨率结构和机制原理。 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的反义调节中涉及的关键中间结构。 这些相互作用也涉及某些mRNA、rRNA和催化RNA结构的正确折叠。 作为基因组逆转录病毒RNA二聚化的重要元件，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“接吻”复合物中特异性相互作用的结构基础：由来自HIV-1基因组RNA中的二聚化起始位点（DIS）的RNA茎-环形成的同二聚体环-环“接吻”复合物和由RNA茎-环形成的环-环“接吻”复合物。来自mRNA CopA和反义CopT阻遏物转录物的环参与R1质粒复制控制;（2）确定环-环“吻”特异性识别的结构基础由蛋白质Rom从ColE 1复制控制系统衍生的复合物。NMR光谱结构和动力学研究将由停流动力学补充，设计用于测定热力学稳定性和缔合的热熔融和凝胶电泳实验/野生型和突变型环-环“接吻”复合物的解离动力学。 新的NMR方法，特别适用于13 C，15 N和/或2 H标记的RNA和RNA-蛋白质复合物的大小（10 kDa - 26 kDa），也将被设计，这将提供更高的精度和灵敏度比现有技术，这将访问全球的长期结构限制目前无法为RNA分子。

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.