HOW DOES A CATALYTIC RNA SWITCH BETWEEN NUCLEASE AND LIGASE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们已经获得了一组晶体学快照的RNA催化使用的活性锤头状核酶的晶体结合化学和物理捕获技术，使我们能够进行时间分辨的单色X射线晶体学分析的核酶的反应坐标。到目前为止，我们已经获得了结构的初始状态的核酶，三个预催化假定的中间构象，和切割形式的RNA的初步结构。总之，这些晶体结构使我们能够构建一个简单的“电影”，描绘似乎伴随着催化的结构变化。第三个前催化中间体偶然地揭示了两个螺旋茎之间的相互作用调节裂解活性。这一观察结果，以及一个更长的野生型序列的出版物，其中广泛的茎I -茎II相互作用被观察到大大加快正向或反向反应速率（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切换是RNA类病毒的生命周期所必需的，其包含锤头状核酶自切割基序。我们最近结晶了这种较大的野生型RNA，并在SSRL获得了两种晶体形式的天然数据集。我们希望现在能解决结构问题。

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.