Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

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Abstract
The decoding region of the E. coli ribosome was localized by affinity immunoelectron microscopy. .**GRAPHIC**. derivatized at the .alpha.-amino end with the dinitrophenyl group, was bound to the ribosomal P [peptidyl] site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work showed that the cross-link occurs between the 5'' anticodon base of the tRNA and a pyrimidine in the 3'' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing 1 covalently attached tRNA per 30S subunit. EM visualization of the antibody attached to the dinitrophenyl group located the position of the 3'' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper 1/3 of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3'' end of the tRNA which is approximately 80 .ANG. from the site of cross-linking. By extrapolating this distance from each of the antigenic sites, it was concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.