Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex

We report here evidence for mutations that break domain-domain functional c ommunication in a synthetase-tRNA complex, Each synthetase is roughly divid ed into two major domains that are paralleled by the two arms of the L-shap ed tRNA structure. The active-site-containing domain interacts with the acc eptor arm of the tRNA. The second domain frequently interacts with the anti codon-containing arm. By an induced-fit mechanism, contacts with the antico don can activate formation of a robust transition state at a site over 70 A ngstrom away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation o f the anticodon-containing full tRNA versus a substrate based on the accept or arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially lin k an anticodon-binding motif to the catalytic domain. The double mutation h ad no effect on interactions with either the isolated acceptor arm or the a nticodon stem-loop. In contrast to interactions with the separate pieces, t he mutant enzyme was severely impaired for binding the native RNA and lost much of its ability to enhance the rate of charging of the full tRNA over t hat of a substrate based on the acceptor arm alone. We propose that these r esidues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.