MAPPING ESCHERICHIA-COLI ELONGATION-FACTOR TU RESIDUES INVOLVED IN BINDING OF AMINOACYL-TRANSFER-RNA

Two residues of Escherichia coil elongation factor Tu involved in bind ing of aminoacyl-tRNA were identified and subjected to mutational anal ysis, Lys-89 and Asn-90 were each replaced by either Ala or Glu. The f our single mutants were denoted K89A, K89E, N90A, and N90E, respective ly. The mutants were characterized with respect to thermal and chemica l stability, GTPase activity, tRNA affinity, and activity in an in vit ro translation assay, Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongat ion factor Tu in complex with aminoacyl-tRNA, which suggested an impor tant role of Lys-89 and Asn-90 in tRNA binding, Furthermore, our resul ts indicate helix B to be an important target site for nucleotide exch ange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to eithe r Ala or Glu were characterized in an in vitro translation assay. Thei r functional roles are discusses in relation to the structure of elong ation factor Tu in complex with aminoacyl-tRNA.