title: Construction of a fully active Cys-less elongation factor Tu: functional role of conserved cysteine 81.
abstract: In order to study the structural and functional requirements of the essential translational GTPase elongation factor (EF) Tu for efficient and accurate ribosome-dependent protein synthesis, construction of a cysteine-free (Cys-less) mutant variant allowing for the site-directed introduction of fluorescent and non-fluorescent labels is of great importance. However, previous reports suggest that a cysteine residue in position 81 of EF-Tu from Escherichia coli is essential for its function. To study the functional role of cysteine 81 and to construct a fully active Cys-less EF-Tu, we have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitutions as well as the isosteric amino acid serine. By characterizing nucleotide binding, EF-Ts interaction, aminoacyl-tRNA binding, and delivery to the ribosome, we demonstrate that only alanine (or cysteine) can be tolerated in this position and that the serine and methionine substitutions significantly impair aminoacyl-tRNA, but not nucleotide binding. Our findings suggest a critical functional role of the amino acid residue in position 81 of EF-Tu with respect to aminoacyl-tRNA binding. Based on structural considerations, we suggest that position 81 indirectly contributes to aminoacyl-tRNA binding through the accurate positioning of helix B.
authors.affiliation: Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada.
authors.forename: Evelina Ines
authors.initials: EI
authors.lastname: De Laurentiis
authors.forename: Fan
authors.initials: F
authors.lastname: Mo
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D010444:Peptide Elongation Factor Tu; D003545:Cysteine