Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.