The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

Abstract

Guanosine 5'-[y-thioltriphosphate (GTP[yS])forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programed ribosome. However, the rate constant for the subsequent hydrolysis of the f-y pyrophosphate bond (3.9 x 10-3 sl at 50C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage ofthis low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, PhetRNAPhe, and GTP['yS] (2.7 x 10-3 s-') and the second-order rate constant for formation ofthis complex (3.3 x 106 M-' s-1). Therefore, the Kd of the complex may be calculated to be 8.2 x 10-10M. An analogous near-cognate complex-with Leu-tRNAI" in place of Phe-tRNA~Ie has been determined by equilibrium methods to have a Kd >1.7 x 10-6 M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and nearcognate ternary complexes with great accuracy. Therefore, its failure to showthis high specificitywith the physiological ternary complexes containing GTP isdue to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination ofspeed and accuracy required in protein biosynthesis.The selection of an aminoacyl-tRNA (AA-tRNA) by a mRNAprogramed ribosome is one ofthe key reactions determining the fidelity of protein biosynthesis which, by current estimates (1, 2), exceeds 99.95%. The mechanism of this selection process has attracted a good deal ofinterest since Hopfield (3) proposed that it exemplified a form ofsubstrate selection known as kinetic proofreading. The selection is thought to occur according to the following scheme: the mRNA-programed ribosome binds a ternary complex ofEFTu, GTP, and the AA-tRNA with rate constant ki, and it either dissociates this ternary complex with rate constant k-1 or hydrolyzes its GTP with rate constant k2. Second, the products EF-Tu-GDP and Pi dissociate from the ribosome, and the AAtRNA may either enter the ribosomal A (acceptor) site with rate constant k3 or dissociate from the ribosome with rate constant k4, depending on the strength of the codon-anticodon interaction (4-6). We have shown previously (6) that errors are made quite frequently in the first recognition step but that they are usually corrected by the second recognition step, which is therefore known as proofreading (3). This paper addresses the question ofwhy the first recognition step-selection and hydrolysis of a ternary complex-is insufficiently accurate to achieve the required fidelity by itself and hence requires subsequent proofreading. Failure to obtain high specificity in the GTPase reaction has generally been attributed to there being only a small difference in free energy between the cog...

tRNA residues evolved to promote translational accuracy

“…A tRNA sequence that is too "stiff" will not be able to access the bent state sufficiently easily to decode its cognate codon, while a tRNA sequence that is too flexible will read near-cognate codons too well. The offsetting needs of speed and accuracy is a common theme in the design of the translational machinery (Thompson and Karim 1982;Wohlgemuth et al 2011;Johansson et al 2012), so it is not surprising that it is critical in dictating the evolution of tRNA sequences.…”

Evolutionary optimization of speed and accuracy of decoding on the ribosome

“…The much lower in vitro error frequency of 10 27 reported recently [63] can now be attributed to the use of extremely low concentrations of free Mg 2þ that destabilized near-cognate codon recognition complexes much more than cognate ones, leading to an artificially high incorporation of cognate relative to near-cognate amino acids [37]. The about 1000-fold difference between the k 22 values imply a large difference in the thermodynamic stability, DDG8, between cognate and near-cognate codonrecognition complexes [29,31,43,64]. However, this large inherent DDG8 of binding does not enter selection as a simple one-step association-dissociation equilibrium.…”

“…The same principles of kinetic partitioning apply for proofreading. Such a selection mechanism allows for rapid translation; however, the maximum of intrinsically possible discrimination is not achieved, a phenomenon known as trade-off between speed and accuracy [64,65].…”

Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

“…(ii) It could be a tightly ribosome-bound translation factor that forms part of the site involved in translational proof reading (18,48); no tightly bound protein of the appropriate size has yet been described. (iii) SUP45+ could encode a translation termination factor, although this is difficult to reconcile with the sup45-induced mistranslation in vitro (43,44) and suppression of presumed missense mutations in vivo (4).…”