Open AccessEscherichia coli elongation‐factor‐Tu mutants with decreased affinity for aminoacyl‐tRNA
Author(s) -
ANDERSEN Carsten,
WIBORG Ove
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18674.x
Subject(s) - ef tu , escherichia coli , mutant , transfer rna , aminoacyl trna , chemistry , elongation , elongation factor , biochemistry , microbiology and biotechnology , biology , ribosome , rna , gene , materials science , ultimate tensile strength , metallurgy
The two evolutionary well‐conserved histidine residues, His66 and His118, of Escherichia coli elongation factor Tu have been subjected to mutational analysis. The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively. His 118 has also been substituted by glutamate, H118E. The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA. Most conspicuously, the tRNA affinity is reduced or almost abolished. k −1 for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type. The half‐lives for the non‐enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively. The K d is about 20‐times higher for H66A compared to wild type. Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.