Structural basis for aminoacylation of cellular modified tRNA by human lysyl-tRNA synthetase.

The average eukaryotic tRNA contains 13 posttranscriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully-modified cellular tRNA by human lysyl-tRNA synthetase (h-LysRS). The tRNA anticodon loop modifications S34 (mcmsU) and R37 (mstA) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNA are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNA aminoacylation are structurally ordered; docking of the 3'-CCA end in the active site cannot proceed until the lysyl-adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS-tRNA complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium towards the 3'-CCA end 'docked' conformation and allosterically enhances h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.