Fig. 2.
The impact of modified stop codons in the 70S A site on binding and releases rates of RFs. (A) Increasing amounts of RF1 were added to ribosomes programmed with UAA (●), UAP (▲), or UPP (♦) stop codons in the A site. The relative changes in fluorescence activity were fit to the equilibrium KD equation (black line; error bars show the SDs from the mean in three independent experiments). (B) In analogy to RF1, RF2 binding kinetics to UAA (●), UAP (▲), or UPP (♦) were analyzed. The total amount of the supplemented RFs is indicated on the x axis. (C and D) Rates of RF1-mediated f[3H]Met release from 70S complexes formed with UAA (●), UAG (■), UAP (▲), or UPG (▼) were assayed under saturating (420 nM RF1) (C) and limiting (14 nM RF1) (D) amounts of RF1 (error bars depict the SDs from the mean in two independent experiments).