Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli.
The Escherichia coli strain D2216 contains a kirromycin-resistant elongation factor Tu [EF-Tu(D2216); Fischer, E., Wolf, H., Hantke K., & Parmeggiani, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4341-4345]. This stain grows much more slowly than wild-type E. coli strains and contains less than half the amount of EF-Tu. On isoelectric focusing, the whole cell lysate of strain D2216 as well as pure, crystalline EF-Tu(D2216) comprises only a single species indistinguishable from wild-type EF-Tu. In poly(uridylic acid)- [poly(U)] directed poly(phenylalanine) synthesis, enzymatic binding of aminoacyl transfer ribonucleic acid to the ribosome, and susceptibility to trypsin digestion, EF-Tu(D2216) behaves similarly to the EF-Tu from wild-type strains. Kirromycin, which increases the sensitivity to trypsinization of wild-type EF-Tu, has no effect on mutant EF-Tu. In poly(U)-directed poly(phenylalanine) synthesis, partially trypsinized EF-Tu(D2216) displays a 7-fold reduction of its kirromycin resistance as compared to the intact EF-Tu(D2216). This is approximately 300 times less sensitive to the antibiotic than wild-type EF-Tu. The EF-Tu(D2216), purified and crystallized, exhibits a guanosine 5'-triphosphatase activity in the absence of any other physiological effector or kirromycin. This activity is not a contaminant, since it can be selectively stimulated by ribosomes and is inactivated by temperature exactly in the same way as the guanosine 5'-diphosphate binding activity of Ef-Tu(D2216). We conclude that, as consequence of the mutation, the catalytic center of EF-Tu(D2216)-dependent guanosine 5'-triphosphate hydrolysis undergoes spontaneous activation.
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D010444:Peptide Elongation Factor Tu; D011072:Poly U; D011728:Pyridones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011728:Pyridones; D012333:RNA, Messenger; D011072:Poly U; D010444:Peptide Elongation Factor Tu; C008536:mocimycin