Poster abstracts
Poster number 40 submitted by Morgan Bauer
An OB-fold Editing Domain in the Human Multi-aminoacyl-tRNA Synthetases Complex Ensures Translational Fidelity
Morgan Bauer (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA. *Bauer.715buckeyemail.osu.edu ), Anna Bibbo (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA.), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA. )
Abstract:
Aminoacyl-tRNA synthetases (ARSs) are universally conserved enzymes that catalyze that attachment of amino acids to their corresponding tRNAs. ARSs are routinely challenged by near-cognate amino acids with a similar size and shape, potentially leading to tRNA mischarging. To maintain translational fidelity, many ARSs encode editing or proofreading domains; free-standing trans-editing domains that hydrolyze misacylated aminoacyl-tRNAs are also encoded in many organisms. Our lab recently discovered a new class of trans-editing enzymes that are defined by an oligonucleotide/oligosaccharide-binding (OB)-fold, a well-characterized nucleic acid-binding beta-barrel structural motif. This unexpected editing activity was first identified in Saccharomyces cerevisiae (Sc) Arc1p and Trypanosoma brucei (Tb) MCP1. Both of these proteins reside in a multi-aminoacyl-tRNA synthetases complex (MSC) that houses multiple ARSs and one or more other cellular factors. Until recently, these OB-fold proteins were believed to act as scaffolding and tRNA binding factors and their role in editing was unknown.
Sequence and structural analyses revealed that human (Hs) AIMP1, a member of the human MSC, contains an OB-fold and conserved residues shared with Sc Arc1p and Tb MCP1. In vitro deacylation assays showed that Hs AIMP1 robustly hydrolyzed Ser- and Gly-tRNAAla, which correspond to common errors by Hs alanyl-tRNA synthetase. Hs AIMP1 showed a substrate preference for smaller amino acids and mutating conserved residues R219 and N212 to alanine significantly reduced deacylation activity. In contrast, mutating conserved residues D172 and M226 had a minimal impact on activity; these residues may be important for AIMP1 structure, dimerization, or other non-enzymatic functions. Mass photometry analysis revealed that Hs AIMP1 forms a dimer in solution. Interestingly, upon addition of tRNA (in the absence or presence of a crosslinking agent), the population shifted to a monomeric protein-tRNA complex. Taken together, these findings support a model in which AIMP1 is not merely a structural component of the human MSC but a bona fide OB-fold editing enzyme that clears misacylated tRNAs.
Keywords: tRNA , translational fidelity , mistranslation