Interaction between the Different Domains of Aminoacyl‐tRNA and the Elongation‐Factor‐Tu · Kirromycin Complex

Abstract

The effect of aminoacyl-tRNA (aa-tRNA) and derived 3'' aminoacylated fragments on the Escherichia coli EF(elongation factor)-Tu GTPase in the presence of kirromycin was studied using 2 systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA1Val 3'' half molecule, and a purification method for short 3'' aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl-adenosine was able to stimulate the EF-Tu .cntdot. kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follows: A-Val .mchlt. C-A-Val < C-C-A-Val < 3'' valyladenosine dodecanucleotide .mchlt. Val-tRNA1Val 3'' half molecule < Val-tRNA1Val. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu .cntdot. kirromycin and the stimulation by aa-tRNA, its 3'' half molecule and even more by its 3'' short fragments. The effect of aa-tRNA and derived 3'' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM; in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, the effect of the C-C-A-aa and the basic activity of the EF-Tu .cntdot. kirromycin GTPase. The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-), arginine was the most active and leucine the least. Thus, the 3'' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, in the 3'' extremity there are critical sequences for the interaction with EF-Tu and other domains of the aa-tRNA molecule are capable of influencing this reaction, 1 of the most important being the region including the T .psi. C loop and stem.