Shape and location of eukaryotic initiation factor eIF‐2 on the 40s ribosomal subunit of rat liver

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Abstract
The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40s-ribosomal-subunit · eIF-2 · GuoPP[CH2]P· Met-tRNA1) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2α, eIF-2β or eIF-2γ. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40s ribosomal subunit in the region between the ‘head’ and the ‘body’ (neck region) of the 40s ribosomal subunit. Within this region, eIF-2α points to the rear side, whereas eIF-2β and eIF-2γ point to the frontal side of the 40s subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4074 × 10−7 cm2/s respectively. From these data, a molar mass of 122.4kg/mol and a dry volume of 147.4nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.