Elongation factor Tu isolated from Escherichia coli mutants altered in TufA and tufB

In a previous paper we described a number of Escherichia coli mutants resistant to the antibiotic kirromycin. These mutants are altered in both tufA and tufB, the genes coding for elongation factor Tu (EF-Tu). We have now isolated EF-Tu in a homogeneous form from the mutant strains and have studied its function in polypeptide synthesis. These EF-Tu preparations were examined in renaturation studies of Qbeta RNA replicase, described in another paper. In order to characterize the factor we have inactivated the tufB gene by insertion of bacteriophage Mu or by an amber mutation. This enabled us to isolate EF-Tu as a single gene product derived from tufA (designated EF-TuA in contrast to the tufB product, which is called EF-TuB). Kirromycin-resistant EF-TuA did not respond to addition of the antibiotic in three assays: [(3)H]GDP exchange with EF-Tu-GDP at 0 degrees C, in vitro translation of poly(U), and kirromycin-induced GTPase activity of EF-Tu. In contrast, wild-type EF-TuA responded normally to the antibiotic in these assays. One of our mutants (LBE 2012) harbors the kirromycin-resistant EF-TuA and an EF-TuB that is able to bind kirromycin. This binding does not cause inhibition of protein synthesis, indicating that EF-TuB from LBE 2012 is unable to reach the ribosome under these conditions. The two types of EF-Tu from this mutant are equal in size but differ by 0.1 pH unit in isoelectric point. In the soluble fractions of LBE 2012 cells they are present in approximately equal amounts. Our results also show that the tufB gene is not necessary for bacterial growth.