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，这些都是许多生物学过程的中心问题。这一建议的广泛目标是利用多维异核核磁共振(核磁共振)波谱和其他生物物理方法，阐明在三个不同和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械原理。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，是参与细菌各种细胞过程反义调控的关键中间结构。这些相互作用也参与了某些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一个可行的靶点。本研究的具体目的是：1)确定两个新的RNA环-环“接吻”复合体中特定作用的结构基础：由HIV-1基因组RNA中的二聚化起始点(DIS)产生的RNA茎环形成的同源二聚体环-环“吻”复合体和由mRNA CopA衍生的RNA茎环形成的环-环“吻”复合体，以及参与R1质粒复制调控的反义Copt抑制子转录本；以及2)确定蛋白质Rom对来自ColE1复制控制系统的环-环“吻”复合体的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得到停流动力学、热熔融和凝胶电泳实验的补充，这些实验旨在分析野生型和突变环-环“接吻”络合物的热力学稳定性和缔合/解离动力学。还将设计新的核磁共振方法，特别适用于13C、15N和/或2H标记的大小(10 kDa-26 kDa)的RNA和RNA-蛋白质复合体，这种方法将提供比现有技术更高的精度和灵敏度，并将获得目前RNA分子无法获得的全球长程结构限制。

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.