Photo‐Induced Protein‐RNA Cross‐Linking in Mammalian 60‐S Ribosomal Subunits

Rat liver 60‐S ribosomal subunits were submitted to increasing doses of radiation (253.7 nm), at 4°C and 25°C, as previously reported for 40‐S subunits. The existence of protein‐RNA cross‐linking was demonstrated by two methods. The first consisted in the separation of protein‐RNA complex; the second was indirect, and took into account alteration either in the electrophoretic mobility of cross‐linked proteins or the separability of 28‐S RNA in a 4 M urea/3 M LiCl buffer. The peptide synthetase activity and the sedimentation characteristics of the particles irradiated at 4°C were well preserved, but at 25°C the large subunits were progressively inactivated and unfolded for doses higher than 2 × 1018 quanta. The dose‐dependent variations of protein cross‐linkage determined by two‐dimensional gel electrophoresis allowed us to distinguish those proteins which reacted at the lowest doses with a first‐order reaction from those which cross‐linked to RNA after a subtle modification of the subunit structure. At 25°C, all proteins became low‐dose reactive. The curve obtained for 28‐S RNA cross‐linkage was similar to that of the total protein moiety, while those obtained for the 5‐S and 5.8‐S RNA (which were parallel) suggest a lower reactivity of these RNAs. As a general rule, proteins from the large subunits were more reactive to RNA than those from the small subunits. This could indicate differences in the organisation of the two subunits.