The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.

No abstract

Specific alterations of the elongation factor Tu (EF‐Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala‐375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF‐Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross‐linked to Lys‐357, also located on domain II but structurally very far from Ala‐375, these data suggest that the replacements alter the relative position of domains I and II. The Ala‐375 replacements also lower the dissociation rates of the binary complexes EF‐Tu.GTP and the binding constants for EF‐Tu.GTP and Phe‐tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly‐222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF‐Tu. Gly‐222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF‐Tu to sustain protein synthesis. It increases the dissociation rate of EF‐Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF‐Tu.GDP does not bind to the ribosome, in contrast to its wild‐type counterpart. A possible explanation is now open for experimental verification.

Abstract-Houseflyhead cholinesterase was purified using the following steps: (1) freeze-drying of flyheads, (2) solubilization of the enzyme by butanol extraction, (3) ammonium sulphate precipitation at pH 7, (4) heat denaturation of proteins in the presence of acetylcholine for protection of the cholinesterase, (5) ammonium sulphate fractionation at pH 7 and at pH 6, (6) calcium phosphate gel absorption and elution, and (7) acetone fractionation.The final preparation, a solution in glass-distilled water, hydrolysed acetylcholine at a rate of 1600 pM/hr/mg of organic matter (157 x purification). It proved fairly stable and was used for studying some properties of the enzyme. The substrate specificity did not change much in the course of purification.The purified enzyme differed from purified bovine cholinesterase in that it hydrolysed butyrylcholine, triacetin, and phenylbutyrate at a much higher rate. The evidence strongly points to one single enzyme being responsible for the hydrolysis of all substrates studied, including butyrylcholine. Inhibition experiments with organophosphates indicate a probable turnover number in acetylcholine hydrolysis of about 100,000. Experiments on the influence of organic solvents showed that 2-3 y0 la-butanol increases the enzymic activity on choline esters about 60%) and that n-butanol, acetone, and ethanol all lower the rate of inhibition by an organophosphorus compound (diazoxon). Agar gel electrophoresis at pH 8.4 showed the cholinesterase to migrate, probably together with other proteins still present in the purified preparation, at a speed which is about 0.9 times the speed of human serum albumin.

We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu in Escherichia coli. T o this aim we have determined the intracellular concentrations of EF-TuA and EF-TUB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild-type cells the expression of fufA and tufB is regulated coordinately. This coordination is not restricted to steady-state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF-TUB from the cell (by insertion of bacteriophage Mu D N A into tufB) nor elevation of the intracellular EF-TUB concentration (by transformation with plasmids harbouring tyfB) has any effect on the expression of tufA. A specific single-site mutation of tufA, however, rendering EF-TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression of tufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild-type cells the EF-Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF-Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA-tgfB transcriptional unit as a template. They show that addition to this system of EF-Tu in concentrations 2 -5 of the endogenous amount results in strong inhibition of EF-Tu synthesis.We hypothesize that EF-Tu acts as an autogenous repressor, inhibiting tufB expression post-transcriptionally.During protein biosynthesis in the bacterial cell, the elongation factor EF-Tu mediates the binding of aminoacyltRNA to the ribosomes [1,2]. It is of interest that the intracellular EF-Tu concentration is approximately equimolar to that of aminoacyl-tRNA [3]. This implies that EF-Tu is one of the most abundant proteins in the bacterial cell and exceeds in concentration the other elongation factors and the ribosomes by a factor of about 10 [3 -61 (and this paper).Another remarkable feature of EF-Tu is its encoding by two unlinked genes, distantly located on the Escherichiu coli linkage map [7]. One of these genes, tufA, is positioned at 73 min and is the promoter-distal gene of the so-called str operon harbouring also the genes coding for the ribosomal proteins S12 and S7, and for the elongation factor C [8]. The other gene, tKfB, lies near 88 min in the rifregion [9] and is cotranscribed with four upstream tRNA genes [lo, 111. The nucleotide sequences of tufA and tufB were found to differ at 13 positions only [12,13] and the corresponding gene products EF-TuA and EF-TUB are identical except for the C-terminal amino acid residue [14,15]. N o functional differences between the two proteins have bee...