The Role of Guanosine Triphosphate Hydrolysis in Elongation Factor Tu-promoted Binding of Aminoacyl Transfer Ribonucleic Acid to Ribosomes

The role of guanosine triphosphate in the polypeptide elongation factor Tu (EF-Tu)-promoted binding of aminoacyl-tRNA to ribosomes has been investigated. In the presence of EF-Tu and GTP, the binding of phenylalanyl-tRNA to the A site of the poly (U)-ribosome complex having an N-acetylphenylalanyl-tRNA prebound to its P site (Complex I) resulted in the formation of N-acetyldiphenylalanyl-tRNA. 5'-Guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP, could substitute for GTP in promoting the binding of phenylalanyl-tRNA to ribosomes in the presence of stoichiometric amount of EF-Tu. However, very little dipeptide formation was observed under this condition. In contrast, when the complex formed with Gpp(CH2)p was incubated at 37° for 5 min and reisolated by centrifugation through 10% sucrose, approximately 50% of the bound radioactivity was recovered as N-acetyldiphenylalanine. This is presumably due to the detachment of Tu-Gpp(CH2)p from ribosomes during reisolation. It was concluded that the role of GTP hydrolysis in this reaction is to remove EF-Tu from ribosomes after the completion of the binding of aminoacyl-tRNA. The release of EF-Tu may be necessary to facilitate the peptidyl transfer and also to initiate the cyclic reutilization of EF-Tu.