Investigating the Catalytic Mechanism of the HDV Ribozyme

HDV核酶催化机制的确定。 丁型肝炎病毒（HDV）是人类病原体B型肝炎的卫星病毒; HDV的共感染加重了B型肝炎的症状。这个基因组含有 病毒复制所必需的核酶序列。尽管我们对 核酶催化在25年来取得了巨大的进步， 尽管发现了这些新的催化剂，但它们的催化机制在很大程度上仍不确定。虽然水晶 许多核酶的结构已经被解决， 许多核酶的结构数据和功能实验之间的联系 包括HDV核酶的系统。在这项工作中，我们将联合收割机核酸 化学与定量酶表征，以开发强大的方法 用于探测HDV的结构、催化和动力学的基本特征 核酶实验将针对核酶领域的三个大问题：（1） 现有的结构是否提供了催化剂的准确表示？ 建筑？(2)核酶如何介导一般的酸/碱催化？(3)如何 核糖磷酸骨架的柔性（动力学）的改变影响功能吗？ 我们期望本文描述的实验不仅将产生对以下方面的基本见解： HDV核酶机制，特别是，但将有普遍的影响， RNA。

Defining the Catalytic Mechanism of the HDV Ribozyme. The hepatitis delta virus (HDV) is a satellite virus of the human pathogen hepatitis B; coinfection with HDV exacerbates the symptoms of hepatitis B. This genome contains the sequence for a ribozyme essential for viral replication. Although our understanding of ribozyme catalysis has advanced enormously in the two and half decades since their discovery, their mechanisms of catalysis remain largely undefined. Although crystal structures for many of these ribozymes have been solved, significant discrepancies between the structural data and functional experiments remain with many ribozyme systems including the HDV ribozyme. In this work, we will combine nucleic acid chemistry with quantitative enzymatic characterization to develop powerful approaches for probing fundamental features of structure, catalysis, and dynamics in the HDV ribozyme. Experiments will be directed at three big questions in the ribozyme field: (1) Do the available structures provide an accurate representation of the catalytic architecture? (2) How does the ribozyme mediate general acid/base catalysis? (3) How do alterations in flexibility (dynamics) of the ribose phosphate backbone affect function? We expect that experiments described herein will not only yield fundamental insights into the HDV ribozyme mechanism, in particular, but will have general implications for all RNAs.