title: Site-directed mutagenesis of Thermus thermophilus elongation factor Tu. Replacement of His85, Asp81 and Arg300.
abstract: His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu.GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-Tu, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.
authors.affiliation: Laboratorium für Biochemie, Universität Bayreuth, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Zeidler
authors.forename: C
authors.initials: C
authors.lastname: Egle
authors.forename: S
authors.initials: S
authors.lastname: Ribeiro
authors.forename: A
authors.initials: A
authors.lastname: Wagner
authors.forename: V
authors.initials: V
authors.lastname: Katunin
authors.forename: R
authors.initials: R
authors.lastname: Kreutzer
authors.forename: M
authors.initials: M
authors.lastname: Rodnina
authors.forename: W
authors.initials: W
authors.lastname: Wintermeyer
authors.forename: M
authors.initials: M
authors.lastname: Sprinzl
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D001224:Aspartic Acid; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012346:RNA, Transfer, Amino Acyl; D001224:Aspartic Acid; D006639:Histidine; D001120:Arginine; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu