STRUCTURAL STUDIES ON RIBOSOMES .2. DENATURATION AND SEDIMENTATION OF RIBOSOMAL SUBUNITS UNFOLDED IN EDTA

The effect of removing Mg2+ from 30- and 50-S ribosomal subunits and ribosomal RNA by the direct addition of EDTA at room temperature has been followed using the analytical ultracentrifuge. 50-S subunits unfolded by a cooperative process, without the dissociation of the protein and RNA moieties, via two discrete, partially unfolded species, to give a 16-S particle. Ribosome-like particles were reformed when the unfolded species were dialysed into magnesium acetate. The unfolding process could be represented as follows:. 50 S ⇌ 35 S ⇌ 21 S ⇌ 16 S. The unfolded ribosomes showed a limited polyelectrolyte behaviour on altering the Na+ concentration or removing Mg2+ and a tendency to specifically refold as the Na+ concentration was increased. By contrast the sedimentation coefficient of 23-S RNA showed progressive changes as Mg2+ was removed with EDTA. No discrete intermediate species were formed. The native 50-S ribosome and the unfolded species had the same total amount of secondary structure and the denaturation behaviour of the 16-S species was the same as 23-S RNA in EDTA. In the unfolded state therefore, the protein did not affect the denaturation behaviour of the RNA. The RNA in the native ribosome was, however, more stable to denaturation than 23-S RNA. This stabilisation was attributed to the tertiary structure of the ribosome. These and other hydrodynamic considerations implied that protein interactions played an important part in maintaining the tertiary structure of the 50-S subunit and suggested that protein was associated with the interhelical regions of the RNA. More limited studies on the 30-S subunits showed that these unfolded to give an 18-S particle without dissociation of the protein and RNA. Under certain conditions the secondary structure of the 18-S species was less than that in the intact ribosome. The thermal denaturation behaviour of the native 30-S ribosome when compared with 16-S RNA showed that, as with the 50-S ribosome, the tertiary structure was stabilised by protein interactions, the protein being associated with the interhelical regions on the RNA. © 1969.