Watkins Rylan R, Bockelman Stella, Vradi Anna, Grabarkewitz Kaylee, Pyun Alexa, Stark Josephine, Wysocki Vicki H, Alfonzo Juan D, Musier-Forsyth Karin
Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, 43220, United States.
Department of Molecular Biology, Cell Biology and Biochemistry, The Brown RNA Center, Brown University, Providence, RI, 02912, United States.
Nucleic Acids Res. 2025 Apr 22;53(8). doi: 10.1093/nar/gkaf328.
Aminoacyl-tRNA synthetases (ARSs) are indispensable for all living organisms and their associated aminoacyl-tRNA editing domains ensure the fidelity of translation. In eukaryotes, ARSs form a multi-aminoacyl-tRNA synthetase complex (MSC), which is assembled together with several nonsynthetase scaffolding proteins. The MSC found in Trypanosoma brucei (Tb) includes two proteins with oligosaccharide/oligonucleotide-binding (OB) folds-MSC-associated protein 1 (MCP1) and MCP2-and one known trans-editing factor, MCP3, an Ala-tRNA deacylase. The activity of MCP1 was unexplored until now. Our study shows that recombinantly-expressed and purified MCP1 also deacylates Ala-tRNAs despite lacking known tRNA-editing domain homology. Domain deletion studies reveal that the OB-fold houses the catalytic pocket and mutation of any one of three conserved OB-fold residues (K326, R331, S335) abolishes activity. Assays with Saccharomyces cerevisiae Arc1p reveal that MCP1's deacylation activity is conserved across organisms. This discovery explains the 3' CCA-end binding activity of this protein family and uncovers an ancient nucleic acid binding domain's unexpected enzymatic function.
氨酰-tRNA合成酶（ARSs）对所有生物来说都是不可或缺的，并且其相关的氨酰-tRNA编辑结构域确保了翻译的准确性。在真核生物中，ARSs形成了一个多氨酰-tRNA合成酶复合体（MSC），该复合体与几种非合成酶支架蛋白组装在一起。在布氏锥虫（Tb）中发现的MSC包括两种具有寡糖/寡核苷酸结合（OB）折叠的蛋白质——MSC相关蛋白1（MCP1）和MCP2——以及一种已知的反式编辑因子MCP3，一种丙氨酸-tRNA脱酰基酶。直到现在，MCP1的活性仍未被研究。我们的研究表明，重组表达并纯化的MCP1尽管缺乏已知的tRNA编辑结构域同源性，但也能使丙氨酸-tRNAs脱酰基。结构域缺失研究表明，OB折叠包含催化口袋，三个保守的OB折叠残基（K326、R331、S335）中的任何一个发生突变都会使活性丧失。对酿酒酵母Arc1p的分析表明，MCP1的脱酰基活性在不同生物中是保守的。这一发现解释了该蛋白家族的3' CCA末端结合活性，并揭示了一个古老核酸结合结构域出人意料的酶功能。
