Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome

Abstract

We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

Keywords: error hot spots; genetic code; kinetics; misreading; protein synthesis.

Fig. 1.

tRNA selection on the ribosome. (A) Kinetic scheme of peptide bond formation on the mRNA programmed ribosome. aa-tRNAs can be rejected during initial selection or at the proofreading step. The two selection steps are separated by hydrolysis of EF-Tu–bound GTP. (B) The efficiency-accuracy trade-off in initial selection was evaluated for different aa-tRNAs reading all codons differing from their fully matched codons by one base change. Asterisk indicates modified base, its position in tRNA anticodon, and its chemical nature. tRNA^Lys data from ref. are included in the article for comparison.

Fig. 2.

Measurements of k_cat/K_m parameters for GTP hydrolysis during cognate or near-cognate codon reading. Time evolution of the level of [³H]GDP in response to [³H]GTP·EF-Tu·Cys-tRNA^Cys binding to 70S ribosomes programmed with cognate codon or near-cognate codons. For the cognate reaction in short time frame (Top Left), data were fitted to a single exponential function. Each near-cognate experiment was performed in parallel with a cognate experiment (in black). The very same ternary complex mixture was here used for both cognate and noncognate reactions, and both curves were jointly fitted with sharing of parameters to increase precision of the measurement (SI Text). In all experiments ribosomes were in excess over ternary complexes, and k_cat/K_m values were calculated from the apparent GTP-hydrolysis rate constant divided by the active ribosome concentration (here, 0.7 µM and 1.8 µM ribosomes were used for cognate and near-cognate reactions, respectively). The decrease in [³H]GDP level in the long time frame is due to spontaneous dissociation of [³H]GDP from EF-Tu followed by its rapid regeneration to [³H]GTP by pyruvate kinase. All experiments were performed in polymix buffer with the addition of 2 mM extra Mg(OAc)₂.

Fig. 3.

The rate-accuracy trade-off. (A) Efficiency of cognate GTP hydrolysis, (k_cat/K_m)^c, for different tRNAs (see B for symbol legend) reading their fully matched codons at varying Mg²⁺ concentration. (B) Efficiency of noncognate GTP hydrolysis, (k_cat/K_m)^nc, for different tRNAs reading single-mismatch codons at varying Mg²⁺ concentration. (C) Efficiency of cognate GTP hydrolysis, (k_cat/K_m)^c, vs. the accuracy [calculated as the ratio (k_cat/K_m)^c/(k_cat/K_m)^nc] for different tRNAs reading single-mismatch codons as indicated in B. In each tRNA misreading case, cognate and noncognate (k_cat/K_m) values were measured at different Mg²⁺ concentrations as shown in A and B. The x intercept gives the maximal accuracy, d, for each misreading case, and the y = 1 line intercept gives the rate constant for association of each cognate tRNA to the ribosome. The complete rate-accuracy trade-off for Glu-tRNA^Glu misreading codon GCA (black triangles and black lines) is plotted in Fig. S1. (D) The rate constant, k_a, for association of different aa-tRNAs in ternary complexes to ribosomes, estimated from the linear dependence between cognate GTP-hydrolysis efficiency and accuracy. Data in A–C represent weighted averages from at least two experiments ± propagated SD. Error bars in D represent the SD estimates from the parameter fitting procedure, where experimental errors, such as in A and B, are used as weights (SI Text). ${\text{tRNA}}_{\text{UUU}}^{\text{Lys}}$ data are from ref. .

Fig. S1.

The rate-accuracy trade-off for Glu-tRNA^Glu misreading codon GCA. Related to Fig. 3C.

Fig. S2.

Maximal accuracy variation dependent on codon position, mismatch identity, and aa-tRNA. Maximal accuracy values, d, for single-mismatch readings by different tRNAs, summarized with respect to mismatch codon position (columns) and mismatch identities (rows for the anticodon bases and colors for the codon bases as indicated in the figure). ${\text{tRNA}}_{\text{UUU}}^{\text{Lys}}$ data are from ref. . For wobble readings (*), see Inset. Errors represent the SDs estimated from the parameter fitting procedure (SI Text, Data Analysis).

Fig. 4.

Comparison between accuracy calculated from in vivo measurement and maximal accuracy of initial selection (d values) for tRNA^Glu misreading its near-cognate codons. Accuracy (black squares) is calculated as the inverse of in vivo error frequency measured by Manickam et al. (36). To compare with our in vitro measurements (d values), in vivo error frequency (f_{in vivo}) for each mistranslated codon was also normalized according to the abundance of tRNA^Glu and the competing cognate tRNA (1) or release factors (for UAA) in E. coli, assuming different tRNAs have similar efficiencies (k_cat/K_m values) for binding to the ribosome in vivo. $f_{nom}=1/A=f_{\text{in\hspace{0.17em}vivo}}(\left[{\text{tRNA}}^{\text{Cognate}}\right]/\left[{\text{tRNA}}^{\text{Glu}}\right])$. d values (red squares) are from Table S1.