ASPARAGINE-135 OF ELONGATION FACTOR-TU IS A CRUCIAL RESIDUE FOR THE FOLDING OF THE GUANINE-NUCLEOTIDE-BINDING POCKET

This work studies the structure-function relationships of Asn135, a re sidue situated in the GTP binding pocket of elongation factor Tu (EF-T u). For this purpose we constructed EF-TuN135D/D138N and assayed its r eactivity towards various purine nucleotides. We found that EF-TuN135D /D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lac k of a productive interaction with isoGTP shows that the Asn135 side-c haindoes not recognize the exocyclic keto group of the guanine base. H owever, EF-TuNl35D/D138N, whose native conformation is stabilized by e ither elongation factor Ts or kirromycin, was able to support the enzy matic binding of aa-tRNA to the ribosome in the absence of any nucleot ide, when in complex with the antibiotic. Taken together, these result s show that Asn135 is important for the correct folding of the nucleot ide binding site and that EF-Tu.kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native c onformation of this site.