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Conformational changes that occur during an RNA-editing adenosine deamination reaction.
H Y Yi-Brunozzi1, O M Stephens, P A Beal
1Department of Chemistry, University of Utah, Salt Lake City, 84112, USA.
The Journal of Biological Chemistry
|August 2, 2001
Summary
Adenosine deaminases acting on RNA (ADARs) selectively edit RNA. The RNA binding domain (RBD) of ADAR2 identifies editing sites and may facilitate base flipping, while the catalytic domain undergoes conformational changes upon RNA binding.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Adenosine deaminases acting on RNA (ADARs) catalyze RNA editing in duplex RNA.
- The precise mechanisms of protein-RNA interactions driving site-selective adenosine deamination remain largely unknown.
- Previous studies suggested a base-flipping mechanism for ADAR2-mediated editing.
Purpose of the Study:
- To elucidate the role of the RNA binding domain (RBD) in ADAR2's site-selective RNA editing.
- To investigate the protein-RNA interactions involved in ADAR2 binding and editing.
- To understand conformational changes in ADAR2 upon duplex RNA binding.
Main Methods:
- Utilized full-length ADAR2 and its isolated RNA binding domain (RBD).
- Employed 2-aminopurine fluorescence assays to monitor substrate interactions.
- Applied hydroxyl radical footprinting to map RNA-RBD interactions.
- Investigated conformational changes using tryptophan fluorescence and acrylamide quenching.
Main Results:
- ADAR2 RBD binding protects the RNA duplex around the editing site, indicating its role in site identification.
- ADAR2 RBD binding induces hypersensitivity to hydrolytic cleavage in adjacent nucleotides, suggesting facilitation of base flipping.
- The catalytic domain of ADAR2 undergoes conformational changes upon duplex RNA binding, with altered tryptophan residue solvent accessibility.
Conclusions:
- The ADAR2 RBD is crucial for identifying specific RNA editing sites.
- The RBD may enhance editing efficiency by increasing the conformational flexibility of the RNA duplex.
- ADAR2 binding to duplex RNA induces significant conformational alterations in the enzyme's catalytic domain.