Specificity and catalysis hardwired at the RNA–protein interface in a translational proofreading enzyme

Proofreading modules of aminoacyl-tRNA synthetases are responsible for enforcing a high fidelity during translation of the genetic code. They use strategically positioned side chains for specifically targeting incorrect aminoacyl-tRNAs. Here, we show that a unique proofreading module possessing a D-aminoacyl-tRNA deacylase fold does not use side chains for imparting specificity or for catalysis, the two hallmark activities of enzymes. We show, using three distinct archaea, that a side-chain-stripped recognition site is fully capable of solving a subtle discrimination problem. While biochemical probing establishes that RNA plays the catalytic role, mechanistic insights from multiple high-resolution snapshots reveal that differential remodelling of the catalytic core at the RNA–peptide interface provides the determinants for correct proofreading activity. The functional crosstalk between RNA and protein elucidated here suggests how primordial enzyme functions could have emerged on RNA–peptide scaffolds before recruitment of specific side chains.