Structural regulation of ribozyme catalysis

描述（由申请人提供）：本文提出的研究的总体目标是了解核酶催化和调节如何工作的基本问题，并将此理解应用于开始设计新的催化特性。我们发现的核酶，调节基因表达的哺乳动物迫使我们了解RNA结构的变化如何使开关从连接（开）状态到切割（关）状态，以及核酶的结构如何产生催化活性在其生物学背景下。使用聚焦机制的X射线晶体学，单分子生物物理学实验和体外进化和选择技术的组合，我们计划回答三组问题，这些问题被制定为提案的三个具体目标。这些实验旨在测试的假设是，RNA本身形成了一个动态的三维结构，不仅调节其整体催化活性，而且还调节RNA切割和RNA连接之间的转换。切割和连接之间的转换对于理解核酶介导的卫星病毒复制和一种新形式的核酶介导的哺乳动物基因调控是绝对关键的。这些特定的目标是：（1）回答全长天然锤头状核酶的活性位点结构如何使其成为酶的问题;（2）理解单个核酶分子如何在所需的核酸酶和连接酶活性之间转换;以及（3）使我们能够设计新的核酶功能，其最终目标是创造新一代潜在的更有效的靶向致病性RNA的体内基于核酶的治疗剂。

DESCRIPTION (provided by applicant): The global objective of the research proposed here is to understand the fundamental question of how ribozyme catalysis and regulation works, and to apply this understanding to begin to engineer new catalytic properties. Our discovery of ribozymes that regulate gene expression in mammals compels us to understand how RNA structural changes enable switching from a ligated (on) state to a cleaved (off) state, and how the ribozyme's structure gives rise to catalytic activity in its biological context. Using a combination of mechanism-focused X-ray crystallography, single molecule biophysics experiments, and in vitro evolution and selection techniques, we plan to answer three sets of questions that are formulated as the three specific aims of the proposal. The hypothesis that these experiments are designed to test is that the RNA itself forms a dynamic three-dimensional structure that regulates not only its overall catalytic activity, but also regulates a switch between RNA cleavage and RNA ligation. The switch between cleavage and ligation is absolutely critical to understanding both ribozyme-mediated satellite virus replication and a new form of ribozyme-mediated mammalian gene regulation. These specific aims are formulated (1) to answer the question of how the active-site structure of the full-length, natural hammerhead ribozyme enables it to be an enzyme; (2) to understand how a single ribozyme molecule can switch between required nuclease and ligase enzyme activities; and (3) to enable us to engineer new ribozyme functionality, with the ultimate goal of creating a new generation of potentially more potent in vivo ribozyme-based therapeutic agents that target pathogenic RNAs.