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

Abstract

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 urea13 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 x 10'' 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.Photochemical studies of Escherichia coli ribosomes 11 -51 have indicated that ultraviolet radiation results in the covalent cross-linkage of ribosomal proteins to rRNA components, and shown that this technique can be used to study specific RNA-protein interaction in intact ribosomes. Recently, we presented evidence indicating that exposure of aqueous solution of rat liver 40-S ribosomal subunits to 253.7-nm radiation also resulted in the covalent cross-linking of proteins to RNA. The identity of these proteins and the kinetics of the cross-linkage reaction were determined. This information was tentatively related to ribosomal structure j 61.The primary objective of this report is to provide a detailed analysis of the effect of 253.7-nm radiation on rat liver 60-S subunits. These subunits are far more complicated than the 40-S ones, since they contain 1.7 times the amount of the total mass of protein and 2.6 times the amount of the total mass of RNA in This manuscript is dedicated to Dr F. Lipmann on the occasion of his 80th birthday.Dejnition. A,," unit, the quantity of material contained in 1 ml o f a solution which has an absorbance of1 at 26Onm. when measured In a 1-cm pathlength cell their structure as compared with small subunits. Furthermore, 60-S subunits contain three rRNA species whereas 40-S subunits contain only one. MATERIALS AND METHODS Subunit PreparationActive ribosomal subunits were prepared as described previously [7], and methylated with formaldehyde and either bo1-01~ Hlhydride or the same unlabelled reagent for 10 min at 20 "C prior to irradiation PI. Ultravio...

Photoincorporation of puromycin into rat liver ribosomes and subunits

“…It is interesting to note that protein and RNA labeling within subunits increased linearly as a function of time, at least up to 30 min of irradiation. As a consequence of all photoincorporation kinetic results, the standard time of photolysis was set to 30 min in all the following experiments, as a compromise between the extent of labeling and the loss of biological activity in poly(U)-directed poly(phenylalanine) synthesis, at most-30% after 30 min of irradiation of either ribosomes or subunits (Reboud et al, 1978(Reboud et al, , 1980aBuisson et al, 1979). Table I summarizes the results obtained as the ionic conditions of the labeling are modified.…”