Binding of Aminoacyl-tRNA. to Ribosomes, Promoted by Elongation Factor Tu Further Studies on the Role of GTP Hydrolysis

The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)-promoted binding of aminoacyl-tRNA to ribosomes has been investigated. In the presence of EF-Tu • and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U) • ribosome complex having N-acetylphenylalanyl-tRNA on its P site. EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'-guanylyl methylenediphosphonate (Gpp(CH1)p), a nonhydrolyzable analog of GTP. The binding of Phe-tRNA. in the former case resulted in the formation of N-acetyldiphenylalanyl-tRNA, while little dipeptide was formed in the latter case. However, when EF-Tu and Gpp(CHi)p bound to the ribosomar complex were released. by centrifugation through 10 % sucrose containing. 0.2 mM GDP, the yield of the dipeptide was correspondingly increased. It was concluded that the role of GTP in this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu-GTP for-ribosomes. The subsequent conversion of EF-Tu-GTP to EF-Tu-GDP, a form of EF-Tu with low affinity for ribosomes as well a