Characterisation of the ribosomal binding site for eukaryotic elongation factor 2 by chemical cross-linking

Abstract

Ribosomal complexes containing elongation factor 2 (EF-2) were formed by incubation of 80 S ribosomes in the presence of EF-2 and the non-hydrolysable GTP analogue Guo PP[CH 2] P. The factor was covalently coupled to the ribosomal proteins located at the factor binding site, by treatment with bifunctional reagents. After isolation of the covalent EF-2 · ribosomal protein complexes, the proteins were labelled with 125I and the introduced covalent links cleaved. The ribosomal proteins were identified by electrophoresis in two independent two-dimensional gel systems, followed by autoradiography. After cross-linking with bis(hydroxysuccinimidyl) tartrate (4 Å between the reactive groups), protein S3 S3 a , S7 and S11 were found as the major ribosomal proteins covalently linked to EF-2. The longer reagent, dimethyl 3,8-diaza-4,7-dioxo-5,6-dihydroxydecanbisimidate (11 Å between the reactive groups), covalently coupled proteins S7, S11, L5, L13, L21, L23, L26, L27a and L32 to EF-2. After cross-linking with dimethyl suberimidate (9 Å between the reactive groups) proteins S3 3 a , S7, S11, L5, L8, L13, L21, L23, L26, L27a, L31 and L32 were identified as belonging to the EF-2-binding site. The results indicate that the ribosomal domain interacting with EF-2 is located on both the small and the large ribosomal subunit close to the subunit interface.