Conformational changes that occur during an RNA-editing adenosine deamination reaction.
ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.
Yi-Brunozzi
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Department of Chemistry, University of Utah, Salt Lake City, 84112, USA.
Stephens
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Beal
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D015075:2-Aminopurine / Q000378:metabolism; D020106:Acrylamide / Q000737:chemistry; D000243:Adenosine Deaminase / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005453:Fluorescence; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D051381:Rats; D014364:Tryptophan / Q000737:chemistry
D017931:DNA Primers; D020106:Acrylamide; D015075:2-Aminopurine; D014364:Tryptophan; D000243:Adenosine Deaminase