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

Abstract

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40s-ribosomalsubunit . eIF-2 . GuoPP[CH2]P . Met-tRNA,) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-24 eIF-28 or elF-2y. 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-2a points to the rear side, whereas eIF-2P and eIF-2y 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 4.74 x lo-' cm'js respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm 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.Eukaryotic initiation factor eIF-2 catalyzes the binding of initiator tRNA to the 40s ribosomal subunit, forming the relatively stable quaternary initiation complex (eIF-2 . GTP . Met-tRNA, . 40s-subunit). Although eIF-2 belongs to the most intensively studied eukaryotic initiation factors (for reviews see [l -5] The observed binding sites for antibodies against each of the three subunits of eIF-2 allow the conclusion that eIF-2 is bound on the interface side in the neck region of the 40s subunit and that the factor molecule has an extension of about 15 nm. The results of hydrodynamic investigations of isolated eIF-2 confirm the conclusion that eIF-2 is of elongated shape.

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF‐2 in the quaternary initiation complex by means of monospecific antibodies

“…Bommer, Central Institute of Molecular Biology, Academy of Sciences of the GDR, Department of Cell Physiology, Robert-Roessle-Strasse 10, DDR-1115 Berlin-Buch, GDR by using polyclonal antibodies against ribosomal proteins, which were shown to be monospecific by immunoblotting and were able to form dimeric immune complexes with 40 S ribosomal subunits. The results demonstrate that ribosomal proteins S3a, S13/16, S19 and $24 are involved in eIF-2 binding, which is in agreement with the observed location of these proteins [7,8] at or near the eIF-2 binding area [9] of the 40 S subunit.…”

“…But our earlier conclusion that protein $6 participates in eIF-2 binding [4] is supported by crosslinking experiments [1,2]. The location of the respective ribosomal proteins on the surface of the 40 S subunit as demonstrated by immunoelectron microscopy [6][7][8] together with the recently observed eIF-2-binding area [9] are shown in a schematic drawing in fig.3. These results clearly support the conclusion drawn from the inhibition experiments with antibodies (figs 1 and 2).…”

“…eIF-2 Fig.3. Three-dimensional model of the 40 S ribosomal subunit demonstrating the location of the antibody-binding sites of ribosomal proteins $2, $3, S3a, S13/16, S17, S19 and $24 (results taken from [6][7][8]) and the location of the elF-2-binding area (results taken from [9]). …”

“…Initiation factor eIF-2 from rat liver was prepared according to Nyghrd et al [12] with some modifications of the procedure described recently [9]. Ribosomal proteins from 40 S subunits were prepared by a procedure including chromatography on CM-cellulose and reversed-phase liquid chromatography [13].…”

“…Most of the proteins shown to be involved in elF-2 binding have been located at the surface of the 40 S ribosomal subunit by immunoelectron microscopy [6][7][8]. However, the location of some of these proteins deviates considerably from the location of elF-2 within quaternary initiation complexes observed recently by the same method [9]. Therefore, we reinvestigated the involvement of ribosomal proteins in the interaction between elF-2 and the 40 S ribosomal subunit…”