A Uniform Response to Mismatches in Codon-Anticodon Complexes Ensures Ribosomal Fidelity

Abstract

Ribosomes take an active part in aminoacyl-tRNA selection by distinguishing correct and incorrect codon-anticodon pairs. Correct codon-anticodon complexes are recognized by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Here, we show by kinetic analysis that single mismatches at any position of the codon-anticodon complex result in slower forward reactions and a uniformly 1000-fold faster dissociation of the tRNA from the ribosome. This suggests that high-fidelity tRNA selection is achieved by a conformational switch of the decoding site between accepting and rejecting modes, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their docking partners at the decoding site. The forward reactions on mismatched codons were particularly sensitive to the disruption of the A-minor interactions with 16S rRNA and determined the variations in the misreading efficiency of near-cognate codons.

Evolutionary optimization of speed and accuracy of decoding on the ribosome

“…This suggests that the network of interactions in the decoding centre [28] has evolved to yield a similar accuracy threshold, independent of the position of mismatches in the codon-anticodon duplex. Limited data obtained with Phe-tRNA Phe , Trp-tRNA Trp and Ala-tRNA Ala suggest that error frequencies are also similar for the near-cognate codons of other aa-tRNAs [30,40,44,45]. Uniform rates of decoding cognate and near-cognate codons presumably reflect the selective pressure that maintains similar translational accuracy for all codons.…”

“…The rates of these reactions are strongly influenced by codon -anticodon interaction [27,30,38,40,41]. On the other hand, the rates of GTP hydrolysis are remarkably similar (within a factor of 3) for different aa-tRNAs on their respective cognate codons; the same is true for the rates of peptide bond formation [40 -44].…”

“…In fact, the rate of GTP hydrolysis by EF-Tu is 2.5 times higher on a cognate codon with a WatsonCrick base pair in the third position compared with a wobble pair [30,46]. Likewise, the rates of GTP hydrolysis on near-cognate codons differ by as much as 20-fold; the same is true for peptide bond formation (figure 2) [30]. Also tRNA modifications play an important role by expanding the repertoire of unconventional base pairs at the third codon position beyond the wobble rules; such codon-anticodon complexes can be treated as 'almost-cognate' and are much less disfavoured than the near-cognate ones [44,47].…”

“…However, aa-tRNAs are about equally distorted on cognate and near-cognate codons, suggesting that, although the distortion of the tRNA structure is necessary for GTPase activation, it is as such not sufficient to account for the discrimination between cognate and near-cognate ternary complexes [36]. The uniformity of mismatch recognition [30] suggests a global response mechanism, which would be consistent with the idea that all conformational changes that occur upon cognate codon recognition, including domain closure of the 30S subunit, distortions of the tRNA and rearrangements in EF-Tu, are essential for the precise positioning of the GTPase centre of EF-Tu at the sarcin-ricin loop, which is required for GTPase activation [48]. Although the tRNA is distorted also in the near-cognate A/T state [36], subtle changes in the orientation of tRNA and EF-Tu could interfere with GTPase activation by preventing A2662 from properly placing His84 into the active site [48].…”

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

“…Given that all individual rate constants are known (5,27), the elemental step (Fig. 1A) can be identified at which tRNA changes the conformation (Fig.…”