Rat liver 60s ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 x lo4 quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5 S RNA and to 28 -5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases TI and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8s RNA, eight with 5 s RNA and among these three are common to both) and have been discussed in the light of all the available data.The proteins and rRNAs in ribosomes are arranged in a specific configuration to form functional domains. In the case of large ribosomal subunits it has been postulated that the 5 s RNA binding area has specific roles in the ribosomal peptidyltransferase and GTPase/ATPase activities, in tRNAbinding and subunit association [I]. As for the large RNA (23 -28 S), recent developments of nucleotide sequence data and phylogenetic sequence comparison show that it contains highly conserved RNA sequences that should be important in peptidyltransferase and GTPase activities [2], most likely with their contacting proteins. The identification of the proteins that interact directly with 5 s RNA and large RNA within the active large subunit is, then, a prerequisite task for understanding the ribosome function. While all published studies agree and show the existence of multiple well-defined 5 s RNA and 23s RNA binding proteins in prokaryotes, there is still controversy about the number and nature of the proteins interacting with eukaryotic 5 s and 28s RNAs (see Discussion).In order to elucidate this question we identified the proteins that were cross-linked to 5s RNA and 28 S RNA within 60s subunits submitted to low doses of short-wave ultraviolet radiations (254nm). A large amount of work done on Escherichia coli ribosomes has shown that protein-RNA cross-linking is induced by these radiations with a high degree of specifity [3-61. Our study, which is a complement to an earlier one [7], allowed us to draw conclusions as to the arrangement of the proteins and rRNAs in the 60s subunits.
MATERIALS AND METHODS
ChemicalsNalz5I(14 Ci/mg) was obtained from New England Nuclear, ribonucleases TI and A were supplied by Worthington.Correspondence to J. P. Reboud, Laboratoire de Biochimie Medicale, Universite Claude Bernard, U.E.R. Lyon-Nord, 43, Boulevard du 11 Novembre 191 8, F-69622 Villeurbanne-Cedex, FranceAbbreviations. RNPEoTA and RNP", the 5 S-RNA -protein complexes that are released from 60 S subunits by EDTA and heating, respectively; SDS, sodium dodecyl sulfate.
Ultraviolet irradiation and analysis of proteins cross-linked to rRNAs60s subunits were prepared from free polysomes, as previously described [S]. They were subjected to reductive methylation prior to irradiation, since we found this procedure effective for diminishing aggregation of irradiated su...