GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu center dot GTP center dot GE2270A complexes

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escheri- chin roll we ch aracterized two mutant EF-Tu species with either G257S or G275A that lead t o high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora r osea to its own antibiotic. Both E. coil mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound con formations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity f or aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE22 70A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of ma g nitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or o ne order, respectively. It thus appears that the resistance mutations do no t prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP- bound conformations the mutant EF-Tus have much less affinity for GE2270A t han wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP GE2270A complex, which predicts direct steric problems between GE 2270A and the mutated G257S or G275A. These mutations may cause a dislocati on of GE2270A in complex with Gm-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the fo llowing novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2 270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligibl e; the former stays at the A-site for subsequent interaction with the pepti dyltransferase centre and the latter two dissociate from the ribosome. (C) 2000 Academic Press.