Fidelity in protein synthesis

Given that tRNAs are aminoacylated with such great accuracy by aminoacyl-tRNA synthetases (10−4–10−5), high fidelity translation then depends on selection of the cognate aminoacyl-tRNA corresponding to the codon presented by the mRNA in the ribosome. Here, the substrates, aminoacyl-tRNAs, are discriminated primarily on the basis of their anticodon sequences. The difference in free energy of binding between the cognate and a non-cognate aminoacyl-tRNA (with two or three mismatches to the codon in the mRNA) is easily large enough to exclude the latter from the ribosome. Discrimination of near-cognate aminoacyl-tRNAs, with only one mismatch between codon and anticodon, with high accuracy, however, is not a trivial problem. There are generally sufficient binding energy differences to allow discrimination between cognate and near-cognate pairings (especially if these differences are sampled several times). But because tRNA selection has the additional constraint of needing to be fast, as translation is rapid and processive, these differences cannot be adequately exploited. Indeed, the rapid rate of translation apparently precludes the establishment of equilibrium between the various tRNAs and the ribosome–mRNA complex, thus calling into action kinetic discrimination mechanisms.The first strategy shown to operate during aminoacyl-tRNA selection has been termed kinetic proofreading. It was long ago realized that, if substrate selection were separated into two distinct phases by an irreversible step (in this case GTP hydrolysis), there would be two opportunities to examine and discard an incorrect aminoacyl-tRNA (Figure 3Figure 3). This means that the binding energy between the ribosome and the ternary complex can be sampled twice and the specificity thus increased. While the idea of having consecutive selective steps is similar to the ‘double-sieve editing’ mechanism discussed above, it is distinguished by the fact that kinetic proofreading applies the same basic selective step twice, whereas editing generally relies on a second distinct site or activity that monitors different properties than the first selective step.Figure 3Detailed kinetic scheme for tRNA selection highlighting the two stages of the process, initial selection and proofreading.The selectivity of the initial selection stage is determined by the difference in rate of GTPase activation (k3) between the cognate and a near-cognate tRNA. The selectivity of the proofreading stage is determined primarily by the difference in rate of accommodation (k5) between the cognate and a near-cognate tRNA. EF-Tu (green) is shown in two different conformations before and after GTP hydrolysis.View Large Image | View Hi-Res Image | Download PowerPoint SlideKinetic proofreading during tRNA selection is made possible by the fact that aminoacyl-tRNAs are delivered to the ribosome in a ternary complex with the GTPase elongation factor Tu (EF-Tu in bacteria, EF1A in eukaryotes) and GTP. In an encounter between ternary complex and the ribosome (initial selection), a cognate ternary complex is more likely to trigger GTP hydrolysis than to dissociate, whereas a near-cognate ternary complex is more likely to dissociate. Simply put, the cognate species partitions forward in the stepwise scheme whereas the near-cognate partitions backward. This initial selection step is followed by the proofreading step where inherent binding differences between codon and anticodon are again sampled. As before, the cognate aminoacyl-tRNA species is more likely to partition forward (and ‘accommodate’ into the A site and participate in peptide bond formation), while the near-cognate aminoacyl-tRNAs are more likely to partition backward (and be rejected from the ribosome).The relative contribution of each of these selective steps, initial selection and proofreading, has been measured in vitro in multiple ways, where overall error rates of ~1 in 450 to 1 in 1600 approach the overall fidelity measured in vivo. In these systems, essentially all non-cognate aminoacyl-tRNAs are rejected during initial selection. Near-cognate aminoacyl-tRNAs, however, can pass through initial selection and trigger GTP hydrolysis with a frequency of ~1 in 30. These sneaky aminoacyl-tRNAs are generally rejected during the second stage, thus increasing selectivity by ~15–45-fold.Interestingly, the maximal theoretical selectivity of kinetic proofreading is not realized here because of the processive nature of translation and associated requirement for speed. To maximize each selective step, the forward rates should be slow enough that differences in dissociation rates can be exploited. Indeed, experimental evidence shows that, when GTP hydrolysis is made extremely slow, the selectivity observed in this initial selection step is substantially increased. It was long ago suggested that ribosome mutations which affect the fidelity of tRNA selection act similarly by increasing or decreasing the rates of individual steps in the selection process.While separating the process into two steps — kinetic proofreading — does provide an advantage during tRNA selection, it is not because differences in dissociation rates between cognate and near-cognate aminoacyl-tRNAs are exploited twice, as previously thought. Rather, during each stage of tRNA selection a second strategy comes into play that introduces a large difference in the rates of two critical forward steps. During initial selection, the rate of GTPase activation (k3) is significantly faster for the cognate than for near-cognate aminoacyl-tRNAs, and during proofreading, there are similar differential rates of accommodation (k5) (Figure 3Figure 3). These differences in forward rates have been attributed to a mechanism historically termed induced fit, which is used by the translation machinery, polymerases and a number of other enzymes. Induced fit refers to the ability of a correct substrate, but not an incorrect one, to cause conformational changes in the enzyme and/or the substrate which have downstream effects on catalysis. During tRNA selection on the ribosome a series of conformational changes induced by binding of the cognate aminoacyl-tRNA, but not a near-cognate one, result in a number of rearrangements in the ribosome and the tRNA itself that result in the kinetic effects discussed above.It appears then that the combination of kinetic proofreading and induced fit in tRNA selection provides a suitable balance between fidelity and rapid elongation rates. If we take the simplest case of kinetic proofreading, where there are no differences in forward rate constants introduced by induced fit — where k3 and k5 are equivalent for both cognate and near-cognate tRNAs — significant discrimination between cognate and near-cognate tRNAs will only be observed when k3 and k5 are very, very slow relative to k–2 and k7. In other words, if tRNA selection were an equilibrium process where the full discrimination potential was extracted from the binding energy, there would be no need for other discriminatory mechanisms. But such a slow step in translation is apparently not compatible with the overall rapid rate of elongation.The addition of induced fit to the process of tRNA selection boosts selectivity when the reaction is constrained to be fast by accelerating the rate of passage of cognate species relative to near-cognate ones. Because forward rates are fast relative to reverse ones, the selectivity of each step is lower than the theoretical maximum allowed by intrinsic energetic differences between cognate and near-cognate tRNAs in the complex. In this case, the energetic cost of inducing conformational changes has little impact on cognate tRNA selection but has substantial detrimental effects on near-cognate tRNA selection thus conferring increased specificity. Effects on forward rates that result from an induced fit mechanism have been shown to be a dominant determinant of fidelity in tRNA selection. A body of experimental data supports this idea by showing that miscoding increases when the differences in GTPase activation and accommodation rates are decreased either by introduction of a mutation in the tRNA body or by addition of antibiotics like paromomycin and streptomycin.We have discussed two general mechanisms used to maintain the high fidelity of protein synthesis (as well as of DNA replication and transcription). The first mechanism is comprised of editing and kinetic proofreading. Although different in detail, both strategies amplify the available discrimination power, determined by differences in free energy of binding, by having more than one selective step. Of these two strategies, editing has an advantage arising from the use of two distinct sites that scrutinize different properties of the substrate. The second mechanism, induced fit, depends on substrate-specific conformational changes that result in selective modulation of forward rate constants, permitting high fidelity discrimination when rapid rates are essential. Such distinct solutions for different enzymes ultimately result from the evolutionary constraints imposed by the diverse requirements for fidelity, speed and efficiency on each molecular problem.