title: Escherichia coli elongation-factor-Tu mutants with decreased affinity for aminoacyl-tRNA.
abstract: 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 The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively. His118 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 Kd 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.
authors.affiliation: Institute of Chemistry, Aarhus University, Denmark.
authors.forename: C
authors.initials: C
authors.lastname: Andersen
authors.forename: O
authors.initials: O
authors.lastname: Wiborg
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D012346:RNA, Transfer, Amino Acyl; D006639:Histidine; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu