Site-directed mutagenesis of Thermus thermophilus elongation factor Tu. Replacement of His85, Asp81 and Arg300

• Published in: European journal of biochemistry Vol. 229; no. 3; pp. 596 - 604
• Main Authors: Zeidler, W, Egle, C, Ribeiro, S, Wagner, A, Katunin, V, Kreutzer, R, Rodnina, M, Wintermeyer, W, Sprinzl, M
• Format: Journal Article
• Language: English
• Published: England 01-05-1995
• Subjects: Amino Acid Sequence; Arginine; Aspartic Acid; Base Sequence; DNA Primers - chemistry; Escherichia coli; Escherichia coli - genetics; Gene Expression; GTP Phosphohydrolase-Linked Elongation Factors - metabolism; Histidine; Hydrolysis; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides - chemistry; Peptide Elongation Factor Tu - chemistry; Peptide Elongation Factor Tu - genetics; Peptide Elongation Factor Tu - physiology; Protein Structure, Tertiary; RNA, Transfer, Amino Acyl - metabolism; Structure-Activity Relationship; Thermus thermophilus; Thermus thermophilus - genetics; Thermus thermophilus - metabolism
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1995.tb20503.x

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.