37] Total reconstitution of 50 S subunits from Escherichia coli ribosomes

Abstract

The 50 S subunit from E. coli ribosomes consists of 34 proteins and two RNA molecules and the 30 S subunit consists of 21 proteins and 16 S RNA. In spite of the large number of ribosomal components, which are all present in one copy per ribosome 2 (except L7/L12:3 or 4 copies), the subunits can be self-assembled in vitro by a technique known as “total reconstitution.” The total reconstitution of the large subunit from E. coli ribosomes requires a two-step incubation of the components (44°, 4 mM Mg 2+ → 50°, 20 mM Mg 2+ ). A conformational change in reconstituted intermediates is the rate-limiting step, and these conformational changes have different ionic and incubation optima. In addition to its importance for assembly studies, the technique of reconstitution is used successfully for the elucidation of the functional role of a number of ribosomal components. The two-step reconstitution leads to particles that are structurally indistinguishable from native 50 S subunits and show 50-100% of the activity of native subunits in various functional tests.