Codon decoding by split-tRNA: revisiting the tRNA selection mechanism

The translation machinery is required to process all codon triplets without exception while maintaining high speed and accuracy, despite orders-of-magnitude differences in cognate pairing stability. For stability-based selection to be efficient, the range of pairing stabilities must be narrowed by raising the lower bound and lowering the upper bound. The constrained structure and intramolecular cooperativity of tRNA complicate understanding of how it modulates codon–anticodon stability and whether it affects selection kinetics beyond codon recognition. To address these questions, we engineered functional split-tRNAs bearing a dangling anticodon in place of the anticodon loop. Our results demonstrate that split-tRNA supports in vitro translation nearly as efficiently as intact synthetic tRNA, challenging the notion that tRNA strain is essential for triggering GTP hydrolysis in response to codon recognition. Using split-tRNA architecture, we found that codon–anticodon stability is likely modulated by the dipole moments of adjacent nucleobases. Our kinetic modeling aligns with a conformational selection mechanism, where the decoding site fluctuates between open and closed states, and the correct codon–anticodon minihelix acts as an allosteric effector that permits its spontaneous closure and stabilizes the closed state. Overall, our data challenge the notion that tRNA is an active player in the selection process.