Download or read book Structural Basis for RNA Editing and Site Selectivity by ADAR2 written by Justin Mclntyre Thomas and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in duplex RNA. Because inosine behaves like guanosine in Watson-Crick base pairing, A-to-I editing may have wide ranging consequences in RNA function. The X-ray crystal structure of the deaminase domain of one member of the ADAR family (ADAR2) was solved over a decade ago, however this structure lacked RNA and provided limited information on how ADARs select, bind and edit adenosines within duplex RNA. This dissertation describes solving of X-ray co-crystal structures of ADAR2’s deaminase domain bound to synthetic duplex RNA and subsequent experiments to define the structural basis of the enzymes sequence preferences. Chapter 2 describes the solved crystal structures of ADAR2 deaminase domain-RNA complexes. The deaminase domain’s RNA binding interface is analyzed and new RNA binding residues are identified. In Chapter 3 the importance of newly identified RNA binding residues is examined through in-vitro biochemical assays. The structural basis for ADAR2’s nearest neighbor sequence preferences are also defined by probing contacts made to the RNA using chemically modified RNA substrates. This information may aid the development of systems to direct A-to-I editing of specific adenosines using ADARs. In Chapter 4, RNA binding experiments using ADAR2 constructs bearing double stranded RNA binding domains (dsRBDs) are carried out with the goal of better understanding how the dsRBDs affect the specificity and behavior of the full length ADAR2. Mobility shift assays, RNA cleavage footprinting assays and electron microscopy all provide evidence that ADAR2 undergoes a specific RNA dependent dimerization. Finally, efforts to obtain high resolution structural data for ADAR2 constructs bearing dsRBDs through X-ray crystallography and Cryo-electron microscopy are discussed