The Kinetics of Ribosomal Peptidyl Transfer Revisited Magnus Johansson, Elli Bouakaz, Martin Lovmar, Måns Ehrenberg  Molecular Cell  Volume 30, Issue 5, Pages 589-598 (June 2008) DOI: 10.1016/j.molcel.2008.04.010 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 A Schematic Representation of Steps that Lead to Peptide Bond Formation on the Ribosome The constant kpep represents the compounded rate constant of all steps subsequent to GTP hydrolysis. Molecular Cell 2008 30, 589-598DOI: (10.1016/j.molcel.2008.04.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Rate of Cognate Dipeptide Formation and GTP Hydrolysis in Polymix Buffer (A) The normalized amount of f[35S]Met-Phe dipeptide formed at 37°C as a function of time for different concentrations of Phe-tRNAPhe containing ternary complex in excess over initiated 70S ribosomes with the cognate Phe codon (UUU) in the A site. (B) The rate of dipeptide formation, estimated from the experiments in (A), plotted versus the concentration of ternary complex. Mean values and standard deviations were estimated from two independent experiments. The smooth curve represents nonlinear fitting of the data to the Michaelis-Menten equation. (Insert) Lineweaver-Burke plot of the data in (B). (C) Time courses of normalized amounts of [3H]GDP formed after saturating concentrations of EF-Tu·Phe-tRNAPhe· [3H]GTP ternary complexes were mixed with initiated 70S ribosomes at different temperatures. Molecular Cell 2008 30, 589-598DOI: (10.1016/j.molcel.2008.04.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Temperature Dependence of kpep and kcat(GTP) during fMet-Phe Dipeptide Formation in Polymix Buffer The rate constants kpep and kcat(GTP) (Table 1) were used to calculate the left side of the transition state equation (Equation 14) for peptidyl transfer and GTP hydrolysis, respectively. These calculated values are here plotted versus the inverse absolute temperature, 1/T. The activation enthalpies can be determined from the slopes, and the activation entropies can be determined from the ordinate intercepts of the straight lines in the figure. Data are represented as mean ± standard deviation. Molecular Cell 2008 30, 589-598DOI: (10.1016/j.molcel.2008.04.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Rate of Near-Cognate Dipeptide Formation in Polymix Buffer The figure displays the rate of f[35S]Met-Phe dipeptide formation per active ribosome at different concentrations of EF-Tu·Phe-tRNAPhe·GTP ternary complexes in excess over the initiated 70S ribosomes with near-cognate Leu codon (CUU) in the A site. Molecular Cell 2008 30, 589-598DOI: (10.1016/j.molcel.2008.04.010) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Rate of Cognate Dipeptide Formation in Tris Buffer (A) The normalized amount of fMet-Phe dipeptide formed at 20°C as a function of time after mixing ternary complexes, containing [3H]Phe-tRNAPhe, with an excess of initiated 70S ribosomes with cognate Phe codon (UUU) in the A site at different concentrations. (B) The rate of fMet-Phe dipeptide formation at 20°C, estimated from experiments with an excess of ternary complexes over ribosomes (■) or the opposite, as in (A), (○), plotted versus the concentration of reactant in excess. Mean values and standard deviations were estimated from two independent experiments. The smooth curves represent nonlinear fitting of the data to the Michaelis-Menten equation. (C) The rate of fMet-Phe dipeptide formation at 37°C for different concentrations of ternary complex in excess over initiated ribosomes. The initiated ribosomes contained either mRNA 022 with weak Shine-Dalgarno or mRNA XR7 with strong Shine-Dalgarno. Data are represented as mean ± standard deviation. The smooth curves represent nonlinear fitting of the data to the Michaelis-Menten equation. Molecular Cell 2008 30, 589-598DOI: (10.1016/j.molcel.2008.04.010) Copyright © 2008 Elsevier Inc. Terms and Conditions