Optimization of speed and accuracy of decoding in translation

Abstract

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k(cat) values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.

Figure 1

Dipeptide formation on a cognate codon. (A) Schematic of the decoding mechanism. Kinetically resolved steps are indicated (Gromadski and Rodnina, 2004a). (B) Dipeptide (fMetPhe) formation in HiFi (open circles) or polymix (closed circles) buffer at 37°C. Increasing amounts of ternary complex (TC=EF-Tu·GTP·Phe-tRNA^Phe) were added to initiation complex with a UUC codon at the A site. (C) Time courses of accommodation and dipeptide formation. Peptide bond formation is shown as consumption of fMet-tRNA^fMet substrate (left y-axis). Accommodation was monitored by the fluorescence decrease after the binding of EF-Tu·GTP·Phe-tRNA^Phe(Prf16/17) to the initiation complex (right y-axis); a.u., arbitrary units.

Figure 2

Dipeptide formation on a near-cognate codon. (A) Time courses of fMetPhe formation under multiple-turnover conditions in polymix buffer. A fixed amount of initiation complex (CUC codon at the A site) was mixed with varying concentrations of EF-Tu·GTP·Phe-tRNA^Phe: 10 (closed circles), 5 (open circles), 2 (closed triangles), and 1 μM (open triangles). (B) Concentration dependence of dipeptide formation under multiple-turnover conditions. (C) Time courses of dipeptide formation under single-turnover conditions measured with limiting concentration of purified ternary complex and varying concentrations of initiation complex: 6 (closed circles), 3 (open circles), 1.2 (closed triangles), and 0.6 μM (open triangles). (D) Rate (closed circles, left y-axis) and amplitude (open circles, right y-axis) dependence of dipeptide formation under single-turnover conditions.

Figure 3

Error frequencies in HiFi and polymix buffers. Error frequencies were determined for polymix (black bars) and HiFi (white bars) buffers. The contribution of initial selection (I) was calculated from the overall error frequency (E) and the measured error frequency of proofreading (P). The calculated error frequency (E_calc) was determined from the ratio of k_cat/K_M values determined for fMetPhe dipeptide formation on near-cognate and cognate codons. The overall error frequency reported by Johansson et al (2008) is shown for comparison (J).

Figure 4

Effect of the Mg²⁺ concentration on dipeptide formation on near-cognate codons. (A) Catalytic efficiency (k_cat/K_M) of dipeptide formation on CUC or CUU codons with (+Mg²⁺) or without (−Mg²⁺) addition of Mg²⁺ to compensate for Mg²⁺-chelating compounds (ATP, 1 mM; GTP, 1 mM; PEP, 10 mM; black bars); the published value (Johansson et al, 2008) obtained under the latter conditions is also indicated (J, grey bar). Inset: Dipeptide formation (k_app) in the presence of uncompensated NTPs and PEP (CUU codon). (B) Effect of Mg²⁺-binding compounds on dipeptide formation. Ternary complex with Phe-tRNA^Phe was added to initiation complex (CUU at the A site) in polymix buffer without further additions (open circles) or after addition of 1 mM ATP/GTP each (closed triangles); 10 mM PEP (open triangles,); or 1 mM ATP/GTP each and 10 mM PEP (open squares). ATP, GTP, and PEP were added with the ternary complex without compensating by Mg²⁺ as described previously (Johansson et al, 2008). As a control, the same amounts of non-compensated ATP, GTP, and PEP were added to the cognate reaction (UUC codon) either with the ternary complex (as in Johansson et al., 2008; closed circles) or with the initiation complex (closed squares). (C) Mg²⁺ dependence of fMetPhe dipeptide formation on cognate (UUC, closed circles) and near-cognate (CUU, open circles) codons. Amplitudes are plotted against the concentration of free Mg²⁺. Inset: time courses of dipeptide formation on the near-cognate CUU codon at increasing concentration of Mg²⁺ (mM): 0.5 (closed diamonds), 1.5 (open squares), 2 (closed squares), 2.5 (open triangles), 3 (closed triangles), 3.5 (open circles) and 5 (closed circles).

Figure 5

Effect of competition on cognate reaction. Rates (k_app) of GTP hydrolysis (closed circles) and dipeptide formation (closed diamonds) were measured on addition of increasing concentrations of competing near- and non-cognate ternary complexes. x-axis, molar excess of near- and non-cognate over cognate ternary complexes.