Limited Proteolysis and Amino Acid Replacements in the Effector Region ofThermus thermophilusElongation Factor Tu

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Abstract
The effector region of the elongation factor Tu (EF‐Tu) from Thermus thermophilus was modified by limited proteolysis or via site‐directed mutagenesis. The biochemical properties of the obtained EF‐Tu variants were investigated with respect to partial reactions of the functional cycle of EF‐Tu. EF‐Tu that was cleaved at the Arg59‐Gly60 peptide bond [EF‐Tu‐(1–59)/EF‐Tu‐(60–405)] bound GDP, EF‐Ts and aminoacyl‐tRNA, had normal intrinsic GTPase activity and was active in poly(U)‐dependent poly(Phe) synthesis. However, the GTPase activity of EF‐Tu‐(1–59)/EF‐Tu‐(60–405) was not stimulated by T. thermophilus 70s ribosomes, and its GTP‐dissociation rate was increased compared with that of intact EF‐Tu. EF‐Tu cleaved at the Lys52‐Ala53 peptide bond has properties similar to EF‐Tu‐(1–59)/EF‐Tu‐(60–405). By means of site‐directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T thermophilus EF‐Tu. These amino acid substitutions did not substantially affect either the affinity of EF‐Tu · GTP for aminoacyl‐tRNA or the interactions with GDP, GTP or EF‐Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome‐induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome‐induced GTPase activity. The results demonstrate that the structure of the effector region of EF‐Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.