Elongation factor Tu resistant to kirromycin in an Esherichia coli mutant altered in both tuf genes

Abstract

A mutant of Escherichia coli is described that displays kirromycin resistance in a cell-free system by virtue of an altered elongation factor Tu (EF-Tu). In poly(U)directed poly(Phe) synthesis the kirromycin resistance of the crystallized enzyme ranged between a factor of 80 and 700, depending on temperature. Similarly, kirromycin-induced EF-Tu GTPase activity uncoupled from ribosomes and aminoacyl-tRNA required correspondingly higher concentrations of the antibiotic. Resistance of EF-Tu to kirromycin is a consequence of a modified enzyme structure as indicated by its altered fingerprint pattern.

Allosteric changes of the guanine nucleotide site of elongation factor EF‐Tu

“…This process proceeds at a faster rate at higher temperatures, but the equilibrium constant is essentially not affected [12], so rate constants of the dissociation reaction at 5°C and the GTPase activity at 37°C can be compared directly. a Originally the EF-Tu produced by D2216 was designated to be a tufB product by genetic evidence [8]. In [16] however, it appeared to be a tufA product from endgroup determination b tufB of LBE2045 was inactivated by insertion of bacteriophage Mu [5,20] 'A' and 'B' refer to tufA and tufB, respectively, the genes encoding EF-Tu; subscripts, S, sensitive; R resistant Each assay (75 ul) contained 50 mM imidazole acetate (pH 7.5), 50 mM KC1, 10 mM MgC12, 0.4 mM GTP, 0.3 mM ATP, 2.6 mM phosphoenylpyruvate, 1 tag pyruvate kinase, 4 vg poly(U), 40 pmol ribosomes, 120 pmol [14C]Phe (900 cpm/pmol), 7 pmol elongation factor G, 88/~g tRNAtntal, 10 pmol elongation factor EF-Ts, 10 pmol EF-Tu and tRNAPhe-synthetase.…”

“…We have isolated mutant E. coli strains resistant to the antibiotic kirromycin [5,8]. This antibiotic inhibits the elongation cycle of protein biosynthesis by preventing the release of EF-Tu from the ribosome after hydrolysis of GTP, thus blocking the ribosome on the messenger RNA [9][10][11].…”

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

“…Mutants of Escherichia coli and Bacillus subtilis selected for resistance to kirromycin have been shown to possess an altered form of EF-Tu [3][4][5]. Acquisition of kirromycin resistance introduces functionally related modifications of the EF-Tu nucleotide-binding site [3,4].…”

A kirromycin resistant elongation factor EF‐Tu from Escherichia coli contains a threonine instead of an alanine residue in position 375

“…Expression of a kirromycin-resistant phenotype requires the alteration of both tufA and tufB [ 10,20,21 ]. Inactivation of tufB by insertion of bacteriophage Mu or by an amber mutation enabled us to isolate single gene products derived from either wild-type tufA (designated EF-TuAs) or kirromycin resistant tufA (designated EF-TuAR).…”