Quantitative study of the interaction of aminoacyl‐tRNA with the a site of Escherichia coli ribosomes

IntroductionPhe-tRNAPhe has a higher affinity to the D site of 70 S ribosomes over wide concentrations of Mgs" and in the absence of elongation factor Tu (EF-Tu) and GTP ]I].If ribosomes were in excess, Phe-tRNAPhe was found exclusively at the D site.This result allowed us to derive the thermodynamic parameters of the D-site binding of aminoacyl-tRNA [2].On the other hand, if high concentrations of Phe-tRNAPhe were used, the A site began to fill after D-site saturation, and quantitative formation of ~he)~-tR~Ap~e occurred f3]_ It made impossible the deter~nat~on of thermodynamic parameters of A-site binding.Here, to avoid this difficulty, we used 70 S ribosomes, where the D sites were initially preoccupied by an anaiogue of peptidyl-tRNA, N-acetyl-f14C]Pl~e-tRNA~~_r~ , exempt from donor activity ]4-6f.In this system Ce measured both association and dissociation rate constants, and the equilibrium binding constants of Phe-tRNAPhe with the vacant A site of 70 S .poly(U) a peptidyl-tRNAEp_rd complex.The latter appeared by 2-3 orders of latitude lower, than the binding constant of Ph~-tRNAFh~ with the D site of 70 S .poly(U) complex.2, Materials md methods 30 S and 50 S ribosomal subunits were isolated from Escherichia coli MRE-600 as in [3].