title: Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.
abstract: Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.
authors.affiliation: Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: O
authors.initials: O
authors.lastname: Wiborg
authors.forename: C
authors.initials: C
authors.lastname: Andersen
authors.forename: C R
authors.initials: CR
authors.lastname: Knudsen
authors.forename: B F
authors.initials: BF
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: Nyborg
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000737:chemistry*; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D012346:RNA, Transfer, Amino Acyl; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu