Rat Liver 60S Ribosomal Subunits Research Articles

Reconstitution of rat liver 60S ribosomal subunits following disassembly by dimethylmaleic anhydride

Modification of 60S ribosomal subunits from rat liver with dimethylmaleic anhydride (60 mumols/ml) is accompanied by release of 35% of the protein. The acidic ribosomal proteins, as well as 9 basic proteins, are selectively liberated from the ribosomal subunits. Reconstitution of the protein-deficient particles with the corresponding split proteins is accompanied by substantial recovery of the original polyphenylalanine synthetic activity. The described reconstitution procedure can be used to investigate the roles played by the released proteins and the functional similarities of proteins from different sources. Hybrid reconstitution of residual ribosomal particles from rat liver or yeast with the corresponding heterologous split proteins produces subunits which have incorporated heterologous proteins but are inactive in polyphenylalanine synthesis.

Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins.

The site of action of a Vero toxin (VT2 or Shiga-like toxin II) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28S ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3'-terminal fragment of 553 nucleotides of 28S rRNA of rat liver 60S ribosomal subunits suggested that an adenine base at position 4324 (A-4324) was absent in toxin-treated 28S rRNA. Further analysis by thin-layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60S ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28S ribosomal RNA.

Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride.

Rat liver 60S ribosomal subunits were treated with dimethylmaleic anhydride, a reagent for protein amino groups, at a 1/15,000 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-2-mediated 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.

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

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 X 10(4) 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 5S RNA and to 28-5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8 S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.

Characterization and properties of a 5S-RNA-protein complex released from heated 60S ribosomal subunits.

When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.

An argument for the existence of a natural complex between protein L5 and 5 S RNA in rat liver 60-S ribosomal subunits

Electrophoresis of the 60-S ribosomal subunits from rat liver in the presence of citrate ions removes the 7 S ribonucleoprotein complex between protein L5 and 5 S RNA though this complex is not released by dialysis or by centrifugation through a sucrose cushion in the same buffer. Using acetate instead of citrate, the subunits remain intact in all cases. On the other hand, in the presence of EDTA, the complex is always released. The poly(U) directed poly-phenylalanine synthesis is correlated in each case with the presence of this complex within the subunits. The melting curves of subunits which have been treated with citrate, acetate or EDTA and then taken back in the buffer in which they were stored suggest that the specific RNA-protein interactions are preserved in the presence of acetate and of citrate but not of EDTA. As a whole, the results support the interpretation that the association of protein L5 and 5 S RNA exists within the active subunits.

Identification of neighboring protein pairs in rat liver 60S ribosomal subunits cross-linked with dimethyl suberimidate or dimethyl 3,3'-dithiobispropionimidate.

Protein-protein neighbors in rat liver 60S ribosomal subunits were investigated by using two bifunctional imidoesters; dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobispropionimidate (DTP). 1. Complexes cross-linked with DMS were separated by two-dimensional acrylamide/urea gel electrophoresis. Each complex in the gel was labeled with 125I (1) and was cleaved into the original monomeric protein constituents of ammonolysis. The products were analyzed by two-dimensional acrylamide/urea gel electrophoresis, followed by radioautography of the stained gel. Eight protein pairs are proposed according to our numbering system (2): L1-L3(L3-L5), L2-L21 (L4-L26), L5-L13 (L7/L7a-L15), L5-L34 L7/L7a-L36), L6-L34 (L8-L36), L6-L32 (L8-L35), L12-L32 (L13/L13a-L35), and L18-L27 (L18-L27/L27a). The designations according to the proposed uniform nomenclature (3) are given in parentheses. 2. Complexes cross-linked with DTP were analyzed by acrylamide/SDS diagonal gel electrophoresis (4), and the molecular weights of the complexes and their components were determined. The monomer components of cross-linked pairs were labeled with 125I in the gel, and identified by two-dimensional acrylamide/urea gel electrophoresis followed by radioautography. Six pairs are proposed; L1-L5 (L3-L7/L7a), L1-L6 (L3-L8). L5-L19 (L7-/L7a-L21/L23/L23a), L13-L15 (L15-L14), L13-L16 (L15-L19), and L23-L27 (L30-L27/L27a). 3. Two pairs which were formed by protein-protein interaction without the use of cross-linking reagents were identified as L2-L4 (L4-L6) and L4-L30 (L6-L29).

Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation

After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultravioletirradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein; these proteins are designated according to the proposed uniform nomenclature. The correlation between that and our nomenclature was reported by McConkey et al. (1979) Mol. Gen. Genet. 169, 1–6. They also confirm the results previously reported (Terao, K., Takahashi, Y. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 230–237).

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 degrees C and 25 degrees C, as previously reported fro 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 degrees C were well preserved, but at 25 degrees C the large subunits were progressively inactivated and unfolded for doses higher than 2 x 10(18) 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 degrees 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 fro 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.

Occurrence of phosphorylated forms of an acidic protein in the large ribosomal subunit of rat liver

An acidic protein from rat liver 60-S ribosomal subunits was selectively extracted with 50% ethanol. It was revealed as three different spots by two-dimensional gel electrophoresis, two of them being attributable to phosphorylated forms since they disappeared after alkaline phosphatase treatment. The relationship between this protein and similar acidic proteins found in eucaryotic cells is discussed.

Studies on the binding of acylaminoacyl-tRNA to rat liver 60S ribosomal subunits and its participation in the peptidyltransferase reaction.

Peptidyltransferase with rat liver 60S subunits can be measured by the reaction between exogenous acylaminoacyl-tRNA and puromycin to form acylaminoacylpuromycin in the presence of 33% methanol, 0.3 M KCl, and 4 mM MgCl2. An assay system has been developed that allows examination of the binding of acetylphenylalanyl-tRNA to the ribosomal subunit "P" site, the transpeptidation of the 60S-bound substrate to puromycin, and the requirements for these individual steps. Binding of acetylphenylalanyl-tRNA to 60S subunits is stimulated several-fold by the addition of methanol, but the extent of binding in alcohol is the same in 60 as in 300 mM KCl containing solutions. Formation of acetylphenylalanyl-puromycin from 60S-acetylphenylalanyl-tRNA complex and puromycin stringently requires alcohol and the initial rate of the reaction is markedly greater at 300 mM KCl than at 60 mM KCl concentrations. Thus, alcohol and high concentrations of monovalent cation affect the reaction of an event subsequent to the binding of substrate to the "P" site. Preincubation of 60S subunits with poly(U), which stimulates the overall peptidyltransferase reaction, does not affect the amount of acetylphenylalanyl-tRNA that is bound to the particles; however, it markedly stimulates the initial rate of the transpeptidation reaction between 60S-acetylphenylalanyl-tRNA complex and puromycin. The codon specificity and the failure to affect binding with poly(U) suggest a role for the polynucleotide in the alignment or stabilization of the acylaminoacyl-tRNA on the "P" site rather than an effect on binding to either of the two particle sites or on the peptidyltransferase "active center." The effect of 40S subunits, which inhibit the overall peptidyltransferase reaction, on the binding of substrate could not be clearly interpreted since all three preparations, 60S subunits, 40S subunits, and combinations of 60S plus 40S particles, appear to bind acetylphenylalanyl-tRNA in the presence of methanol. However, the initial rate of peptide bond formation is several times greater with 60S-acetylphenylalanyl-tRNA complex than with 60S plus 40S particles containing bound acetylphenylalanyl-tRNA and the addition of 40S subunits to preformed 60S-acetylphenylalanyl-tRNA complex during the transpeptidation phase of the reaction in methanol does not affect the rate of peptide bond formation. Thus, 40S subunits seem to inhibit peptidyltransferase by forming less reactive particles in aqueous solutions. Contd.

Characterization of the peptidyltransferase reaction catalyzed by rat liver 60S ribosomal subunits.

Characterization of the peptidyltransferase reaction catalyzed by rat liver 60S ribosomal subunits.