STRUCTURE AND FUNCTION OF RNA-MODIFYING PROTEINS

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。对RNA中的碱基进行修饰已被认为是各种生物基因组中编码的蛋白质多样化的重要机制。许多脱氨酶已被鉴定为催化tRNA、mRNA、甚至病毒RNA中的核苷酸碱基的直接变化。这些蛋白质是产生抗体多样性、对入侵病毒的固有细胞反应以及在翻译过程中引入tRNA简并以识别具有单一tRNA的多个密码子所必需的。目前的研究重点是研究腺苷脱氨酶修饰RNA中特定位点的化学机制。我们目前的研究重点是TADA，这是一种来自细菌的酶，它在特定tRNA的反密码子位置的摆动位置引入肌苷。该方法是利用X射线结晶学产生这些酶与靶向RNA结合的新结构。本实验旨在通过将蛋白质与代表反应途径上不同步骤的RNA中间体结晶来可视化该蛋白质是如何识别和进行RNA的化学修饰的。这些结果将对作用于DNA和RNA的其他密切相关的脱氨酶的RNA识别和催化作用产生影响。

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.