Figure 2.

Determination of the optimal length of the mRNA for fast rates and large fluorescence change

(A) Schematic representation of the peptide elongation cycle on the ribosome starting from the 70S IC. The process includes two peptide bond formation steps and one translocation step driven by EF-G. The di- and tripeptide formation experiments were conducted in quench-flow, where an elongation factor mix containing the respective TCs (5 µM) was rapidly mixed with the 70S IC (0.5 µM) containing mRNA and fMet-tRNA^fMet in the P site. By fitting the kinetic data as described in the Materials and Methods, the mean times of the first peptide bond formation (τ_p1), second peptide bond formation (τ_p2) and tripeptide formation (τ_tripeptide) were determined. Both τ_p1 and τ_p2 include time starting from binding of TC and entire EF-Tu cycle. The mean time of a full translocation reaction (τ_{translocation}) was calculated as [τ_tripeptide– (τ_p1+ τ_p2)]. In parallel, the kinetics of translocation starting from the 70S IC was followed in stopped-flow, where the fluorescence change of the 3ʹ pyrene labelled mRNAs was monitored in real time. The mean time τ_fluor, indicated total time of all events starting from the 70S IC up to and including mRNA movement. The mean time for mRNA movement, τ_{mRNA move}, was determined by subtracting τ_p1 from τ_fluor. (B) Kinetics of the first peptide bond formation and (C) tripeptide formation in quench-flow starting from the 70S IC with the mRNAs as indicated.(D) Kinetics of the second peptide bond formation starting from the post-translocation complex with fMet-Phe-tRNA^Phe in P site. (E) Kinetics of EF-G mediated mRNA movement during translocation in a stopped-flow starting from the 70S IC, monitored by the changes in pyrene fluorescence (343 nm excitation, 360 nm long-pass filter) with the mRNAs.(F) Bar diagram for direct comparison of the mRNAs for the mean times of different steps of elongation. The three stacked bars indicate τ_tripeptide obtained by quench-flow with clear demarcations for τ_p1 (grey), τ_{translocation} (pink/red) and τ_p2 (blue). For fluorescent mRNAs τ_{translocation} (pink/red) is divided into τ_{mRNA mov} (pink) and τ_{post mRNA move} (red). For mRNAs without dye entire τ_{translocation} is in pink. The stopped-flow based mean times τ_fluor are presented by the green bars. The error bars represent standard deviation.