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Effects of mutation glycine 222 aspartic acid on the functions of elongation factor tu



Effects of mutation glycine 222 aspartic acid on the functions of elongation factor tu



Biochemistry 26(7): 2047-2054



We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding of EF-Tu. The resulting EF-TuBo .cntdot. kirromycin and EF-TuAr .cntdot. EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu .cntdot. GTP being slightly faster. Protection of aminoacyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo .cntdot. GTP .cntdot. aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome .cntdot. mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations. Whereas the intrinsic GTPase (the activity found in the absence of any effector) of EF-TuBo is the same as for wild-type EF-Tu, aa-tRNA and ribosomes are incapable of stimulating the GTPase activity of EF-TuBo in the absence of mRNA. Kirromycin stimulates the GTPase of wild-type EF-Tu and EF-TuBo to the same extent with and without aa-tRNA, while ribosomes, normally the most important effectors of the reaction, are unable to enhance the EF-TuBo .cntdot. kirromycin GTPase. These results indicate that the mutation Gly-222 .fwdarw. Asp causes an anomalous interaction of EF-TuBo with the ribosome in both the absence and the presence of aa-tRNA. This alteration increases the initial energetic constraints and makes the presence of mRNA and high Mg2+ concentration prerequisites for a productive interaction of the EF-TuBo ternary complex with the ribosome.

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