Accessibility of RNA and Protein in the Ribosome. Investigation by Hydrogen Exchange and Solvent Perturbation

Conditions have been found which allow the separation of protein and RNA from Escherichia coli 70-S ribosomes while inhibiting hydrogen exchange in both. Thus the location of masked exchangeable hydrogens can be determined. Tritiated ribosomes were allowed to exchange for a period, at the end of which 25% of the tritium atoms per ribosomes were retained. It was found that at least 80% of them were in the protein. This value corresponds to 60% of the total exchangeable protein hydrogens. Since dissociation of the subunits is not accompanied by detectable conformational changes in the proteins, it is concluded that a large proportion of the ribosomal protein, but little of the RNA, is inaccessible to solvent and therefore buried in the interior of the particle. The presence of slowly-exchanging hydrogens in the ribosomal protein was confirmed by observations of deuterium exchange by infrared spectroscopy. Solvent perturbation of the ultraviolet absorption band of ribosomes was observed, using deuterium oxide as perturbant. A Perturbation-difference spectrum indicating accessible RNA on the 70-S particle was obtained. On dissociation of the subunits the perturbation increases. Separated 50-S and 30-S subunits exhibited difference spectra, which were almost unaffected by magnesium concentration over wide limits, suggesting that the dissociation is accompanied, irrespective of magnesium concentration, by some general loosening of the structure, as also infereed from earlier work on tritium exchange of subunits. The available evidence concerning the distribution of RNA and proteins between the surface and the interior of the ribsome is briefly discussed.