The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S].
Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) 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 programmed ribosome. However, the rate constant for the subsequent hydrolysis of the beta-gamma pyrophosphate bond (3.9 X 10(-3) s-1 at 5 degrees C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage of this low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, Phe-tRNAPhe, and GTP[gamma S] (2.7 X 10(-3) s-1) and the second-order rate constant for formation of this complex (3.3 X 10(6) M-1 s-1). Therefore, the Kd of the complex may be calculated to be 8.2 X 10(-10) M. An analogous near-cognate complex with Leu-tRNA2Leu in place of Phe-tRNAPhe has been determined by equilibrium methods to have a Kd greater than 1.7 X 10(-6) M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and near-cognate ternary complexes with great accuracy. Therefore, its failure to show this high specificity with the physiological ternary complexes containing GTP is due 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 of speed and accuracy required in protein biosynthesis.
D005075:Biological Evolution; D004926:Escherichia coli; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D010445:Peptide Elongation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D013873:Thionucleotides; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate