STRUCTURE AND FUNCTION OF RNA-MODIFYING PROTEINS

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。在RNA中引入碱基修饰已被确定为使在各种生物体的基因组中编码的蛋白质多样化的重要机制。已经鉴定了许多脱氨酶，其催化tRNA、mRNA甚至病毒RNA中的核碱基的直接变化。这些蛋白质是产生抗体多样性、对入侵病毒的先天细胞反应以及引入tRNA简并性以在翻译期间用单个tRNA识别多个密码子所必需的。腺苷脱氨酶修饰RNA特定位点的化学机制是目前研究的重点。我们目前的研究集中在TadA上，这是一种来自细菌的酶，可以在特定tRNA的反密码子位置的摆动位置引入肌苷。该方法是使用X射线晶体学产生与靶RNA结合的这些酶的新结构。目前的实验被设计为允许可视化该蛋白质如何识别和进行RNA的化学修饰，通过用代表反应途径沿着不同步骤的RNA中间体结晶该蛋白质。这些结果将对其他作用于DNA和RNA的密切相关的脱氨酶的RNA识别和催化产生影响。

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. Introducing modifications to bases in RNA has been identified as an important mechanism for diversification of proteins encoded in the genomes of various organisms. Numerous deaminases have been identified that catalyze direct changes to nucleobases in tRNA, mRNA, and even viral RNA. Such proteins are required for generating antibody diversity, innate cellular responses to invading viruses, as well as introducing tRNA degeneracy for recognition of multiple codons with a single tRNA during translation. The focus of the current research is to study the chemical mechanism of adenosine deaminases that modify specific sites in RNA. We are focusing our current studies on TadA, an enzyme from bacteria that introduces inosines at the wobble position at the anticodon position of a specific tRNA. The approach is to generate novel structures of these enzymes bound to target RNA using X-ray crystallography. Present experiments are designed to allow the visualization of how this protein recognizes and carries out the chemical modification of RNA by crystallizing the protein with RNA intermediates representing distinct steps along the reaction pathway. These results will have implications for RNA recognition and catalysis by other closely related deaminases that act on DNA and RNA.