Functional split-tRNA: a new perspective on codon decoding mechanism

The translation machinery must rapidly and accurately process all codon triplets despite large differences in the stability of codon:anticodon duplexes. The constrained structure and intramolecular cooperativity of tRNA complicate understanding how its structural elements influence the thermodynamics and kinetics of the selection process. Specifically, it remains unclear whether codon:anticodon complex stability controls kinetics of tRNA selection beyond the codon recognition step. To address this we engineered fully functional split-tRNAs with a dangling anticodon triplet instead of an anticodon loop. Using this tool, we demonstrated that codon-anticodon complex stability is primarily influenced by the dipole moments of adjacent nucleobases and does not control the rate of GTP hydrolysis by EF-Tu. We conclude that the codon-anticodon minihelix functions as a passive steric gate of decoding site closure.