An Escherichia coli mutant with an altered elongation factor Tu

Abstract

A thermosensitive mutant of E. coli has been isolated that is unable to replicate the bacteriophage MS2 at 42° but permits phage production at 37°. Thermal inactivation studies of the supernatant enzymes show that this mutant contains a factor essential for the polymerization of phenylalanine from phenylalanyl tRNA that at 50° is more rapidly inactivated than the corresponding wild type factor. The elongation factor Tu (EF Tu) was isolated and purified to apparent homogeneity, as the EF Tu GDP complex, both from mutant and wild type cells. Addition of purified wild type EF Tu GDP to reaction mixtures fully restored the activity of thermally inactivated mutant supernatants. These experiments excluded EF Ts as the thermolabile factor involved. Similar inactivation studies, dealing with the purified factors and performed in reaction mixtures that were not supplemented with GDP, revealed that the half life of mutant EF Tu GDP at 50° was 1.5 min, that of the wild type factor 6 min. Addition of GDP (10 μM) to the medium reduced the inactivation rate of both wild type and mutant factor and also the difference in inactivation kinetics. Besides the altered elongation factor Tu, the mutant still contains a second mutation affecting the glutaminyl tRNA synthetase.