Ribosomal Subunit Exchange: Analysis in Vitro

The ribosomes repeatedly undergo subunit exchange, by dissociation into their subunits and reformation from a pool of free subunits that continuously recycle through ribosomes. This chapter describes an in vitro assay to study the mechanism of ribosomal subunit exchange and its role in protein synthesis. The assay is capable of measuring a single cycle of ribosome dissociation and reformation from subunits. The ribosomal subunit exchange can be demonstrated in vivo by centrifugal analysis of the density distribution of ribosomes and ribosomal subunits following transfer of a culture uniformly labeled with heavy isotopes to a medium containing only light isotopes. Hybrid ribosomes originating in vivo display essentially the same patterns. These particles may be separated into two species by density-gradient equilibrium centrifugation, if they are not resolved in sedimentation velocity analysis. A sensitive method of measuring small differences in the extent of ribosomal subunit exchange is to label two heavy cell cultures with different radioisotopes.