HOW DOES A CATALYTIC RNA SWITCH BETWEEN NUCLEASE AND LIGASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们使用活性锤头状核酶的晶体结合化学和物理捕获技术获得了一组RNA催化的结晶学快照，这使得我们能够对核酶的反应坐标进行时间分辨的单色X射线结晶学分析。到目前为止，我们已经获得了核酶的初始状态、三个催化前假定的中间构象和RNA裂解形式的初步结构。总而言之，这些晶体结构使我们能够构建一部简单的“电影”，描述伴随催化而出现的结构变化。第三个催化前中间体偶然地揭示了两个螺旋茎之间的相互作用调节切割活性。这一观察结果，以及一个更长的野生型序列的发表，其中观察到广泛的Stem I-Stem II相互作用，极大地加快了正向或反向反应速度(分别)，迫使我们试图了解这种核酸酶-连接酶转换的结构基础。核酸酶和连接酶活性之间的转换是RNA类病毒生命周期中所必需的，它包含了锤头状核酶自切割基序。我们最近已经结晶了这个更大的野生型RNA，并在SSRL获得了两种晶型的原始数据集。我们现在希望解决这个结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have obtained a set of crystallographic snapshots of RNA catalysis using crystals of an active hammerhead ribozyme in conjunction with chemical and physical trapping techniques that have enabled us to conduct time-resolved monochromatic x-ray crystallographic analyses of the ribozyme's reaction coordinate. To date, we have obtained structures of the initial state of the ribozyme, three pre-catalytic presumed intermediate conformations, and a preliminary structure of the cleaved form of the RNA. Together, these crystal structures have allowed us to construct a simple "movie" depicting the structural changes that appear to accompany catalysis. The third pre-catalytic intermediate fortuitously revealed that interactions between two helical stems regulate cleavage activity. This observation, as well as publication of a longer, wild-type sequence in which extensive Stem I - Stem II interactions are observed to greatly accelerate either the forward or the reverse reaction rates (separately), compel us to try to understand the structural basis of this nuclease-ligase switching. The switch between nuclease and ligase activity is required in the life cycles of RNA viroids that incorporate the hammerhead ribozyme self-cleaving motif. We have recently crystallized this larger, wild-type RNA and have obtained native data sets on two crystal forms at SSRL. We hope now to solve the structure.