Ribosomal Subunits Of Rat Liver Research Articles

Shape and location of eukaryotic initiation factor eIF‐2 on the 40s ribosomal subunit of rat liver

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.

Studies on interaction of 5 S RNA with ribosomal proteins

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl 2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and — to a lesser extent — L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.

TRNA binding modifies the properties of the small ribosomal subunit of rat liver.

The binding of a specific tRNA (acylated or not) to the 40S subunits in the presence of the proper codon was shown to produce two striking effects on the subunits. First, the subunits were no longer able to dimerize at low ionic strength. Second, they became fully resistant to 1.25 M LiCl treatment: bound tRNA prevented subunit inactivation as measured by polyphenylalanine synthesis; it also prevented large sedimentation changes of subunits and ribosomal protein release induced by LiCl. The number of protected proteins far exceeded that of the proteins crosslinked with tRNA after irradiation at 254 nm (A.M. REBOUD, S. DUBOST and J.P. REBOUD (1983) FEBS Lett. 158, 285-288). These results strongly suggest that tRNA binding induces modifications of rRNA-protein interactions in large domains of the subunits. A weak interaction of tRNA with the 40S subunit was demonstrated in the absence of the codon.

5.3 S RNA is a discrete cleavage product from the 5′-terminus of 18 S RNA of rat liver ribosomes

A 5.3 S RNA species observed in urea-gel electrophoretic analysis of the RNA of the small ribosomal subunit of rat liver has been identified from its sequence as the 5′-terminal 133–134 base fragment of 18 S RNA. Presumably it is cleaved by an endogenous endonuclease when the ribosomal subunits are dissociated, because it usually is not observed in 18 S RNA obtained by direct extraction of cells or tissues.

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.

TRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.

Identification of neighbouring protein pairs in the rat liver 40-s ribosomal subunits cross-linked with dimethyl suberimidate

1. (1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. 2. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. 3. (3) The following dimers were proposed from these results: S3-S12 (S3 or S3a-S11), S4-S12 (S3b-S11), S5-S7 (S4-S6), S5-S22 (S4-S23 or S24), S6-S8 (S5-S7), S8-S16 (S7-S18), S17-S21 (S16-S19) and S22A-S22B (S23-S24), designated according to our numbering system [1]. The designations according to the proposed uniform nomenclature [2] are described in parenthesis.

Studies on native ribosomal subunits from rat liver. Evidence for a low molecular weight ribosome dissociation factor

Extracts prepared from native ribosomal subunits of rat liver with high salt (0.5 m KCl) solutions contain dissociation factor activity; they dissociate 80 S ribosomes and prevent the reassociation of ribosomal subunits. One of the activities is a high molecular weight (500,000–700,000) protein composed of about 10 polypeptides (eIF-3 H). A low molecular weight activity (eIF-3 L) is resolved from the heavy dissociation factor by glycerol gradient centrifugation. Both activities catalyze dissociation and anti-association. Gel electrophoresis of complexes between ribosomal subunits and eIF-3 L, in sodium dodecyl sulfate, suggests that the low molecular weight dissociation factor contains a polypeptide or polypeptides of 51,000 molecular weight. Comparison of the electrophoretic patterns does not reveal any common polypeptides in the two dissociation factor preparations.

Size and shape of isolated proteins of the small ribosomal subunit of rat liver

Size and shape of isolated proteins of the small ribosomal subunit of rat liver

25] Preparation of a ribosome dissociation factor from native ribosomal subunits of rat liver

25] Preparation of a ribosome dissociation factor from native ribosomal subunits of rat liver

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.

All factors required for protein synthesis are retained on heparin bound to Sepharose.

1. Postmitochondrial supernatants of rabbit reticulocyte lysates were chromatographed on heparin bound to Sepharose 4B, and the fraction retained on affinity columns was separated by subsequent gel filtration on Sepharose 4B into three fractions, two of them active in protein synthesis. 2. The heavier fraction sedimented at 40S and contained more than 10% RNA. This consisted predominantly of a 12S component, with smaller amounts of the 9S and 4S RNA species. The lighter fraction (18-20S) was composed of proteins with less than 1% RNA. 3. Different enzymic activities were associated with these fractions. 4. In the presence of both fractions, efficient translation took place on combined ribosomal subunits of rat liver with added cofactors. Globin messenger ribonucleoprotein stimulated this translation 5-6-fold. 5. Relatively large complexes of all factors required for protein synthesis are apparently isolated from reticulocytes by affinity chromatography on heparin-Sepharose 4B. Such complexes may occur naturally in the cytoplasm of mammalian cells.

Turnover of rat liver 28 S ribosomal RNA Nicking as the initial step of degradation

The large ribosomal subunits of rat liver contain small amount of minor RNA components which migrate through polyacrylamide gels between the two major rRNAs, and some 28 S rRNA molecules contain hidden nicks which can be revealed by heat treatment. The minor RNA components were isolated, labeled in vitro with 125I, and hybridized with nucleolar DNA from rat liver. The hybridization-competition experiments with 28 S RNA established that the minor RNA components and 28 S rRNA have a common DNA origin. When RNA components were isolated from rats that had received [ 14C]orotate and [ 3H]orotate, 1 week and 16 h, respectively, before sacrifice, the 14C : 3H ratio was the highest in the minor components, followed by the nicked RNA, and the intact 28 S rRNA showed the lowest ratio. We also found that partial hepatectomy decreases and the actinomycin d-treatment increases the amount of the minor RNA components as well as the nicked 28 S RNA. From these observations, we conclude that the initial step of degradation of 28 S rRNA in vivo is the nick formation and that the nicked RNA subsequently gives rise to the minor components.

Studies on proteins of animal ribosomes XXIV. Localisation of proteins in ribosomal subunits of rat liver studied by chemical substitution with p-nitrophenyl acetate and methyl acetimidate

The reaction of [ 3H] p-nitrophenyl acetate (NPA) or [ 14C]methyl acetimidate (MAI) with amino groups of ribosomal proteins from the rate has been studied. A comparison has been made between the reactivity of the proteins in situ in the ribosomal subunit with that of isolated protein mixtures. In the small subunit reactivity compared with the protein mixture was only 10–65% in the case of NPA but 45 to more than 100% in the case of MAI. In the large subunit reactivity to MAI was 10–60% that of the isolated protein mixture. This suggests that the large subunit has a denser structure than the small one. In agreement with earlier experiments with iodoacetamide the proteins S2, 5, 7, 8, 10 and 13 of the small subunit and L15, 17, 20, 24, 25, 27, 29, 33, 34, 35 and 38 in the large subunit are quite accessible while proteins S9, 14, 19, 20, 24, 25, 27, 29 and 30 of the small subunit and L1, 7, 8, 10, 11, 19, 28, 31 and 32 of the large one are relatively inaccessible.

Effect of ribonuclease T1 on ribosomal subunits of rat liver.

The accessibility of 28S RNA within the ribosomal subunits to ribonuclease T1 was studied, in comparing results obtained after enzyme treatment of compact, K+ deficient 60S subunits and of EDTA-treated 60S subunits. RNA, extracted from the subunits, using a mixture of sodium dodecyl sulfate and phenol was analyzed on sucrose gradients. The RNA from active subunits was only degraded in high enzyme concentrations. In the K+ deficient subunits, RNA is more accessible since it breaks down into 6 well-defined fragments, sedimenting between 4S and 18.5S. Within the EDTA-subunits, there is no more protection of the RNA. In fact, it is degraded by weak enzyme concentrations, as is the free 28S RNA, giving heterogeneous fragments. Comparison of the melting curves of subunits and free 28S RNA showed that it is only in EDTA subunits that proteins do not stabilize the secondary structure of RNA. In the case of 40S subunits, the action of ribonuclease T1 combines with the action of the endogenous nuclease which makes the degradation process more difficult to analyze.

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm

Heterogeneity among the free small ribosomal subunits of rat liver was studied. Newly made free small subunits differ from the bulk of the free subunits in the following ways. (1) The newly made free small subunits sediment slightly faster than most free small subunits and form a band at slightly lower densities. (2) Mature free small subunits, prepared under conditions which minimize the amount of loosely bound protein, readily dimerize in solutions of low ionic strength. Little dimerization is found with newly formed subunits. This permits the separation of fractions enriched in newly formed subunits. (3) The fraction enriched in newly formed subunits contains a distinctive additional protein as compared the most of the free small subunits.

Minor components of rat liver ribosomal RNA as possible intermediates in the degradation of 28S RNA in vivo effects of partial hepatectomy and starvation on their relative amounts.

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48 × 105 to 11.4 × 105, represented 10 to 13% of the total high-molecular-weight ribosomal RNA.It was elucidated that they were not artifacts arose during the cell homogenization, sub-cellular fractionation, RNA preparation and gel electrophoresis.Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo.In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.

Conformational changes of large ribosomal subunits of rat liver, induced by some monovalent cations

Li + and Cs +, which selectively inactivated the 60-S ribosomal subunits of rat liver at a relatively low concentration (0.3 M), were shown to induce structural changes in these subunits. Thus, much more ribosomal proteins were found to be exposed to a protein reagent (procion brilliant blue) in large subunits prepared using Li + and Cs + than in subunits prepared with other monovalent cations. Also, Li + and Cs + (0.5 M) released most of the 5-S RNA from the large subunits, but that was not the case with the other monovalent cations. Both observations are probably related to a considerable unfolding of the large subunits.