Rat liver 60s ribosomal subunits were treated with dimethylmaleic anhydride, a reagent for protein amino groups, at a 1/15000 mol/mol ratio. This caused the dissociation of specific proteins, which were separated from the 56s residual core particles by centrifugation and identified by two-dimensional gel electrophoresis. The core particles lacking 30% of the total proteins retained most of the initial activity measured by the puromycin reaction but only small percentages of activities measured by polyphenylalanine synthesis, elongation-factor-2(EF-2)-dependent GTP hydrolysis and EF-Zmediated GDP binding. Upon reconstitution, the complementary amount of split proteins was incorporated into ribosomal particles, which had almost the same catalytic activities and biophysical properties (density, sedimentation coefficient and capability to reassociate to 40s subunits) as the original subunits.
Incubation of 60 S ribosomal subunits with the ricin A chain reduced their stability during heat treatment. The toxin shifted the thermal denaturation curve of the subunits towards lower temperatures, in a similar way to that produced by the decrease in Mgz+ concentration. A brief heating (3 min at 57"C), which did not affect control subunit activity, enhanced protein synthesis inhibition of the toxin-treated subunits that released more 5 S RNA, in the form of nucleoprotein complex(es) with protein L5 and phosphoproteins PlP2 (RNP& than did heated control subunits [(1984) Eur. J. Biochem, 143,303-3071. No nuclease activity tested on 60 S subunits and purified 5 S and 5.8 S RNA was found associated with the toxin. The results suggest that the toxin induced a limited conformational change of the 60 S subunit, which destabilized the interaction between RNPH and the rest of the subunit. Ricin (Eucaryote)Ribosomal subunit RNase SSRNA
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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.