Investigating the Catalytic Mechanism of the HDV Ribozyme

确定HDV核酶的催化机理。 丁型肝炎病毒(HDV)是人类病原体乙肝的卫星病毒； 与HDV的混合感染会加剧乙肝的症状。这个基因组包含 核酶对病毒复制至关重要的核酶序列。虽然我们对此的理解 核酶催化在过去的25年里取得了巨大的进步。 尽管已有发现，但它们的催化机制在很大程度上仍不清楚。虽然水晶 其中许多核酶的结构已经被解决，显著的差异 在结构数据和功能实验之间留下了许多核酶 包括HDV核酶在内的系统。在这项工作中，我们将结合核酸 化学与定量酶特性相结合，开发强有力的方法 用于探索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) 可用的结构是否提供了催化剂的准确表示 架构？(2)核酶如何介导一般的酸/碱催化？(3)如何 磷酸核糖骨架柔韧性(动力学)的改变会影响功能吗？ 我们预计，这里描述的实验不仅将产生对 尤其是HDV核酶机制，但将对所有 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.