An Escherichia coli mutant with an altered elongation factor Tu.
A thermosensitive mutant of Escherichia coli has been isolated that is unable to replicate the bacteriophage MS2 at 42 degrees but permits phage production at 37 degrees . 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 degrees 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 degrees was 1.5 min, that of the wild-type factor 6 min. Addition of GDP (10muM) 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 skill contains a second mutation affecting the glutaminyl-tRNA synthetase.
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002250:Carbon Radioisotopes; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D010445:Peptide Elongation Factors / Q000302:isolation & purification*; D010649:Phenylalanine; D012329:RNA, Bacterial / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D010649:Phenylalanine; D012343:RNA, Transfer