Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tu(mt), show distinct differences in their affinit ies for guanine nucleotides and in their interactions with elongation facto r Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu bin ds GDP much more tightly than EF-Tu(mt). This difference does not reside in domain I alone but is regulated by interactions with domains II and III. A U the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRN A although some had an increased or decreased activity in this assay. The a ctivity of E. coli EF-Tu but not of EF-Tu(mt) is stimulated by E. coli EF-T s. The presence of any one of the domains of EF-Tu(mt) in the prokaryotic f actor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tu(mt) allowed the mitochondrial factor to interact with bacterial EF-Ts. This observatio n indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Ts-mt interacts wit h all of the chimeras produced. However, with the exception of domain III e xchanges, it inhibits the activities of the chimeras indicating that it cou ld not be productively released to allow formation of the ternary complex. The unique ability of EF-Tu(mt) to promote binding of mitochondrial Phe-tRN A(Phc) to the A-sire of the ribosome resides in domains I and IT. These stu dies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains. (C) 1999 Elsevier S cience B.V. All rights reserved.