Photo‐induced protein cross‐linking to 5S RNA and 28–5.8S RNA within rat‐liver 60S ribosomal subunits

Abstract

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...

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear Overhauser enhancement

“…biosynthesis (Rohl & Nierhans, 1982;Brewer et al, 1983). Since its discovery (Rossett & Monier, 1963), 5S RNA has been studied intensively via genetic, enzymatic, chemical, and physical techniques in an effort to unravel its structure and the nature of its interactions with ribosomal proteins (Vandenberghe et al, 1985;Paleologue et al, 1985;Bohm et al, 1985;Kjems et al, 1985;Eigen et al, 1985; Curtiss & 0006-2960/86/0425-5117$01.50/0 © 1986 American Chemical Society Vournakis, 1984; Ohama et al, 1984;Delihas et al, 1984;Pieler et al, 1984;Nazar & Wildeman, 1983;Nazar et al, 1982).…”

RNA PROTEIN CROSSLINKS INTRODUCED INTO E. coli RIBOSOMES BY USE OF THE INTRINSIC PROBE 4‐THIOURIDINE

“…Even at the moderate doses used, the ribosome activity tested in protein synthesis reactions decreased strongly with apparently no other proteins being crosslinked. However, other groups were able to crosslink many other proteins in other types of ribosomes (Paleologue et al, 1985) or with E. coli ribosome and tRNA ribosome complexes, yielding much information of structural interest (Turchinsky et al, 1978). Since the doses used (2-10 x lo5 quantdparticle) are in the same range, the different results reported could be accounted for in part by the different sensitivities of the detection methods: 1251 labeling (Turchinsky et al, 1978), silver staining (Paleologue et al, 1985) and Coomassie blue staining (Moller and Brimacombe, 1975;Ehresmann et al, 1980) and overall in vivo (32P)RNA labeling for detection of crosslinked oligonucleotides.…”