A rapid and sensitive assay for the detection of eukaryotic ribosome dissociation factors.

A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNA_f^met to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg²⁺ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg²⁺ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg²⁺ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg²⁺ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [³⁵S]Met-tRNA_f^met in the presence of wheat germ eIF-2. The [³⁵S]Met-tRNA_f^met bound to the 40 S subunits is readily detected by its retention on a Millipore filter.