Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection

Abstract

Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg 2 concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.fidelity | rate-accuracy trade-off | ribosome | protein synthesis | elongation T ranslation of the ancient and universal genetic code into protein on ribosomes requires precise mRNA decoding by aminoacyl-tRNAs (aa-tRNAs) and rapid formation of nascent peptide chains (1, 2). Codon reading by aa-tRNAs ultimately relies on the specificity of cognate in relation to noncognate codon-anticodon interactions, but two ribosome-dependent specificity enhancements greatly improve mRNA decoding (3). Firstly, bases A1492 and A1493 in the 16S rRNA of the 30S subunit have stereospecific, A-minor interactions with the first two codon-anticodon base pairs but not with the third (4-6), suggesting higher frequency of misreading of the third codon base than of the first two bases (3), in line with the wobble hypothesis (7). Secondly, the ribosome enhances the accuracy of codon reading by a twostep mechanism in which initial codon selection by a tRNA is followed by a proofreading step (8-11). That is, the ternary complex consisting of aa-tRNA, elongation factor Tu (EF-Tu), and GTP is, when cognate, activated for GTP hydrolysis on EF-Tu with high probability, whereas a noncognate ternary complex is likely to dissociate from the ribosome before GTP hydrolysis. After GTP hydrolysis on EF-Tu, a cognate aa-tRNA is selected for peptidyl transfer with high probability, whereas a noncognate aa-tRNA is likely to dissociate from the ribosome in a proofreading step before peptidyl transfer (8, 9) (Fig. 1).In spite of the central role that is played by ribosome aided reading of the genetic code in all areas of biology, very little is known about the basal parameters that provide the boundary conditions for rapid and accurate mRNA translation into protein.Most important among these parameters is the maximal possible discrimination between a cognate and a noncognate codon-anticodon interaction: the "d value." It defines the upper limit of the current single step accuracy, A, by which any aa-tRNA can separate its cognate from a specific noncognate codon. When the c...

Two proofreading steps amplify the accuracy of genetic code translation

“…The existence of two distinct proofreading steps may appear surprising, because the accuracy of initial codon selection by ternary complex normally is remarkably high (22,31). Therefore, we suggest that two-step proofreading has evolved to neutralize the deleterious effects of a small number of distinct error hot spots for initial codon selection (31) as observed in vitro (19) and in vivo (18).…”

“…The concentration of free Mg 2+ ions affects the accuracy of initial codon selection, I, in mRNA translation by decreasing the rate constant for dissociation of ternary complex from its precodon recognition state (21,22). Here, we first studied the effect of Mg 2+ concentration on the proofreading parameter, F, by which Glu-tRNA Glu favors its cognate GAA codon in relation to its near-cognate GAU, GGA and GAC codons.…”

“…2A), GGA, and GAC in the A site of ribosomes with initiator fMet-tRNA fMet in the P site. In each case, the ratio between cognate and near-cognate k cat /K m values estimated the initial selection accuracy, I (22). We also determined the k cat /K m values for fMet-Glu-tRNA Glu formation in each one of these four cases and thereby obtained the total accuracy, A = I × F, of peptide bond formation with Glu-tRNA Glu reading its cognate GAA codon versus the near-cognate GAU (Fig.…”

“…1). The simplistic idea was that, because the accuracy of initial selection of codons by aa-tRNA in ternary complex with EF-Tu·GTP decreases sharply with increasing Mg 2+ concentration (22), such a high Mg 2+ sensitivity could be typical also of a first proofreading step in which aa-tRNA in ternary complex with EF-Tu·GDP is discarded. Furthermore, because the accuracy of factor-free codon selection has comparatively low sensitivity to Mg 2+ concentration (Fig.…”

Self-Referential Encoding on Modules of Anticodon Pairs—Roots of the Biological Flow System

“…The primacy of the central base [29] is reflected in some regularities that are recognized in the code matrix since the time of its deciphering about half a century ago, especially the correlation of the most hydrophobic amino acids coinciding with the most hydrophobic central purine A, which is the first column. Evidence from the synthetase-tRNA interactions [34] (Table 3) indicates that the situation is highly complex.…”