J.Martien de Graaf

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

The peptide chain elongation factor EF-Tu from Escherichia coli is an allosteric protein with multiple activities in the bacterial cell. Besides its function in protein synthesis [ 1,2], it acts as a subunit of the enzyme Q/3 RNA replicase [3]. Its extremely high intracellular abundance [4,5] and its peripheral association with the inner membrane of the E. eoli cell [4] have led to speculations about an additional structural role for this remarkable protein [6,7]. Two unlinked genes coding for EF-Tu have been identified on the E. coli chromosome [8-10]. One designated tufA maps at 72 rain, the other, designated tufB, at 88 min. Their nucleotide sequences have recently been elucidated [ 11,12]. The primary structures of the corresponding gene products designated EF-TuA and EF-TuB, respectively, were thus found to be identical, except for the C-terminal amino acid residue (Gly or Ser). This is in accordance with the directly determined primary structure of the mixture of the two proteins [ 13,14]. EF-Tu interacts with a great number of ligands and in some cases this interaction has been correlated with conformational changes of the polypeptide chain [2,15,16]. To study the relation between the structure and function of this multifunctional and allosteric protein, we have isolated a number ofE. eoli mutants with an altered EF-Tu. We took advantage of the fact that the antibiotic kirromycin binds to the factor in an 1:1 molar ratio [17]. This binding to EF-Tu • GDP induces an EF-Tu • GTP-like conformation which prevents the release of the factor from the ribosome, thus blocking protein synthesis [ 15,18,19]. 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). Both protein species have the same relative molecular mass (Mr) and isoelectric point [22]. EF-TuA R has a strongly reduced affinity for kirromycin, but its function in polypeptide synthesis appeared to be normal [ 17,22,23 ]. On the other hand, if EF-TuA R replaces its wild-type counterpart in 033 replicase the enzyme is rapidly inactivated and displays an apparent increase in template specificity [24]. Here we show that Ala-375 of EF-TuA S is substituted by Thr in EF-TuA R. The importance of this substitution for structure and function of the enzyme is discussed.

Divergent effects of fluoroaluminates on the peptide chain elongation factors EF-Tu and EF-G as members of the GTPase superfamily

Fluoroaluminates are thought to mimic the γ‐phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP‐binding G‐proteins. Here we show they do inhibit the ribosome‐stimulated GTPase activity of EF‐G from Escherichia coli via the formation of a stable complex with EF‐G·GDP and ribosomes. In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF‐Tu. Interestingly, in the absence of ribosomes both EF‐Tu and EF‐G remain totally unaffected by fluoroaluminates. For members of the GTPase superfamily such differential effects have not been described before.

The primary structure of the coat protein of alfalfa mosaic virus (strain 425)

Abstract The complete amino acid sequence of the coat protein of alfalfa mosaic virus (AMV 425) is presented. The protein chain consists of 220 residues corresponding to a calculated molecular weight of 24,253. Some rough predictions are given concerning the secondary structure.

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.