Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Translocation kinetics. PyC-labeled E. coli Cys-tRNA^Cys (2 μM) was loaded onto the A site of the programmed E. coli ribosome (1 μM) and rapidly mixed with EF-G-GTP (1 μM). The fluorescence change of the translocation time course of the tRNA from A to P site is shown, displaying the rapid formation of an intermediate (k ₁ = 19.0 ± 1.7 s⁻¹) from the PRE complex and a slower conversion of the intermediate to the POST complex (k ₂ = 3.6 ± 2.0 s⁻¹).