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Supplemental Text
For the analysis of rapid kinetic data obtained on the cognate UUU codon, the concept of net rate constants was used (Cleland, 1975). The suitability of this approach for the analysis of data obtained under pre-steady-state conditions, provided the backward reactions of all steps except for the first one are negligible, was shown earlier (Bilgin et al., 1992; Savelsbergh et al., 2003). Rate constants were calculated for the model depicted in Fig. 2 (see text), assuming k-2»0. Derivation of the concentration dependencies of the apparent rates (kapp) of steps 2 and 3 (Cleland, 1975) yields:
Apparent rates were estimated by exponential fitting of time courses obtained by quench-flow or stopped-flow measurements (Fersht, 1998). The experiments were performed at fixed concentration of ternary complex and increasing concentrations of ribosome complexes which were always in excess over the ternary complex (pseudo-first-order conditions). Concentration dependence of kapp2 was obtained monitoring the fluorescence of proflavin-labeled tRNA. From the saturation of the hyperbolic fit, k2 equals to 190±20 s-1 (eqn. 1). The KM value obtained from the data of Fig. 3C, 1.6±0.4 µM, was in agreement with that calculated from k1=140 µM-1s-1, k-1=85 s-1 (Fig. 3B), and k2=190±20 s-1 (Fig. 3C), 2.0±0.6 µM (eqn. 1). Concentration dependence of kapp3 was obtained from time courses of GTP hydrolysis (Fig. 4E). At saturation, kapp3=110±25 s-1; from k2=190±20 s-1 and eqn. 2, k3=260±80 s-1.
The net-rate approach is not suitable for the analysis of the data obtained on the near-cognate codon, CUC, because k-2 is comparable to k2. However, as k3=0.4 is much smaller than k2 and k-2, the reaction can be treated as being close to equilibrium at the stage of codon recognition, and the formalism for the analysis of the concentration dependence of a two-step reaction can be used (Bernasconi, 1976; Fersht, 1998) (see text).
The measured values of k2 and k-2 were verified using an EF-Tu mutant that was deficient in GTP hydrolysis, EF-Tu(H84A). Three steps take place prior to GTP hydrolysis: initial binding of ternary complex to the ribosome, codon recognition, and GTPase activation (Daviter et al., 2003). Fluorescence increase of proflavin was monitored upon interaction of EF-Tu(His84A)·GTP·Phe-tRNAPhe(Prf16/17) with CUC-programmed ribosomes as described in Materials and Methods (not shown). The GTPase activation step does not lead to Prf fluorescence changes (Rodnina et al., 1995) and is therefore not observed in these experiments. Furthermore, the GTPase activation step was shown to be reversible, with an internal equilibrium close to 1 (Daviter et al., 2003). At increasing ribosome concentrations, the apparent rate constant of the reaction increased hyperbolically and saturated at 220±20 s-1 (not shown), indicating that the fluorescence change reports a step following the bimolecular binding step, i.e. codon recognition. That also the amplitude of the fluorescence change increased supports the notion that the reactions are reversible (if any of the reactions were irreversible, identical amplitudes were expected). The concentration dependence of the amplitudes yields an overall Kd=0.4±0.1 µM for the three steps which is given by K1·K2·K3, where K1, K2, and K3 are equilibrium dissociation constants of initial binding, codon recognition, and GTPase activation, respectively. The rate constants of initial binding, k1 and k-1, were not affected significantly by the mutation of EF-Tu (Daviter et al., 2003). Therefore, values of k1 and k-1 from Table 1 were used, resulting in K1=0.6. Assuming K3=1 (see above), K2=k-2/k2 was calculated to 0.66. At saturation, kapp=k2+k-2 (Fersht, 1998). From these two relationships, k2=140±30 s-1 and k-2=80±20 s-1 were obtained for the near-cognate ternary complex, in agreement with the data of Table 1.
The values for kcat/KM of GTP hydrolysis were calculated using the previously described approach (Pape et al., 1999) according to the equation:
For the cognate codon, k-2»0, and the expression simplifies to
where kcat=k2 and KM=(k-1+k2)/k1.
For the near-cognate codon, k3»0, and the expression simplifies to
where kcat=k3 and KM=(k-1·k-2)/(k1·k2).