Ribosomal Protein Fraction Research Articles

Effects of the inhibition of ribosomal-RNA synthesis on the synthesis of ribosomal structural proteins in regenerating rat liver.

Employing regenerating rat liver, the metabolic coordination between ribosomal RNA and ribosomal proteins was investigated. 1 When the synthesis of ribosomal RNA was selectively inhibited by a low dose of actinomycin D, the incorporation of labeled amino acid not only into protein components of cytoplasmic ribosomes but also into those of nucleolar 60-S particles were markedly decreased. The inhibition was rather selective. 2 The ribosomal protein fraction (Fraction I) was purified from the acid-soluble proteins of total cell homogenate by CM-cellulose column chromatography. Fraction I showed many bands with the same mobilities as those of ribosomal structural proteins on acrylamide gel electrophoresis. Actinomycin D treatment for 5 min to 1 h inhibited the incorporation of labelled amino acids into Fraction I and its subfractions on acrylamide gel electrophoresis. The maximal level of inhibition was obtained by the actinomycin D treatment for 20 min. 3 Acrylamide gel electrophoresis of Fraction I, prepared from nascent protein fractions of rat liver ribosomes pulse-labeled with amino acids, showed that the incorporation into nascent ribosomal proteins was inhibited by the actinomycin D treatment for 1 h. 4 It is indicated that the biosynthesis of the bulk of ribosomal proteins itself may be coordinated with that of ribosomal RNA in regenerating rat liver.

Biogenesis of ribosomes: Free ribosomal protein pools in Escherichia coli

Proteins from ribosomal subunits (30 s and 50 s) have been fractionated into split (SP-30 and SP-50) and core (core-30 and eore-50) proteins. Antisera prepared in rabbit against them are shown to be highly group-specific as judged by the Ouchterlony (1967) double diffusion test and by precipitin reaction in solution. Various parameters which influence the immuno-precipitation of these proteins by specific antisera have been investigated. It is demonstrated that under controlled conditions this provides a sensitive and reliable method for characterization and quantitative estimation of free ribosomal proteins. The technique has been successfully applied in the investigations of various properties of free ribosomal protein pools existing in Escherichia coli. It is concluded that free ribosomal proteins in E. coli may constitute 8 to 14% of total soluble proteins under different growth conditions. Relative pool sizes of four classes of proteins expressed as a percentage of the total soluble proteins in bacteria grown in L broth (doubling period, 30 min) are estimated to be core-30, 2.1; core-50, 3.4; SP-30, 3.6; SP-50, 5.0. From the studies on bacteria grown in different media (doubling period, 30, 42 and 60 min), we further conclude that the amount of free proteins increases with the growth rate so as to constitute a constant fraction (7 to 9%) of total ribosomal proteins. Relative pool-sizes corresponding to four classes of ribosomal proteins, however, remain unaltered by different growth rate. Studies on the time-course of incorporation of labelled amino acids show that the level of radioactivity (specific activity) in free ribosomal protein pools reaches a saturation value in 2 to 4 minutes with an average half-life of about 50 seconds. Kinetic curves (specific activity versus time) for free and bound ribosomal proteins are found to be compatible with a precursor-product relationship. Pulseand-chase experiments further suggested that there is no significant breakdown of proteins in pools and that they are quantitatively transferred to mature ribosomes. Relative pool-sizes and kinetic behaviour of free ribosomal proteins indicate that these proteins are synthesized in a co-ordinate manner. Various possible mechanisms for a co-ordinate synthesis of ribosomal RNA and proteins at the level of subunits and at the level of whole ribosomes are discussed.

Studies on the metabolism of ribosomal structural proteins of regenerating rat liver. Sites of biosynthesis of structural proteins of large subunit and of their assembly with RNA moiety.

In order to elucidate the site of the synthesis of ribosomal proteins and of their assembly with ribosomal RNA in liver cells, labeling experiments were performed in vivo, employing regenerating rat liver and the following results were obtained. 1 The relative synthetic rates of 28-S ribosomal RNA and ribosomal proteins at various time after partial hepatectomy, which were expressed as the ratios to those of the normal rat liver, ran parallel with each other and had maximal values, more than 10-times higher than those of normal rats, at 12 h after partial hepatectomy. 2 Disc electrophoresis of the acid-soluble proteins extracted from nucleolar detergent extract or 60-S particles by the procedures of Narayan and Birnstiel [1] revealed that the protein fraction contained most species of structural proteins of cytoplasmic ribosomes. Furthermore, the profiles of radioactivity on disc electrophoresis of the mixture of acid-soluble proteins of nucleolar 60-S particles and those of the large subunit of cytoplasmic ribosomes which were labeled with 3H and 14C-labeled amino acids, respectively, showed that most of the peaks of 3H and 14C radioactivities coincided with each other. The results may indicate the similarities between the protein moieties of these two particles. 3 The time courses of the incorporation of [3H]leucine into acid-soluble proteins of the nucleolar detergent extract and into the basic structural proteins of the cytoplasmic ribosomes suggested the precursor nature of proteins of the nucleolar extracts to structural proteins of the cytoplasmic ribosomes. Similarly the time-course study on the incorporation of [14C]orotic acid into RNA moieties and that of [3H]leucine into protein moieties of both nucleolar 60-S particles and the large subunit of the cytoplasmic ribosomes, indicated that 60-S nucleolar particles may be the precursor to the large subunit of cytoplasmic ribosomes. 4 The nascent ribosomal structural protein fraction, which was purified from the nascent protein fraction of regenerating-liver ribosomes labeled with [3H]leucine was subjected to disc electrophoresis. The radioactive proteins were distributed in the region of ribosomal structural proteins in addition to some amounts of radioactivity in more fast-moving regions. The time course of the incorporation of [3H]leucine into this nascent ribosomal proteins showed a sharp increase after the injection, reached a maximal value after 5 min and thereafter decreased rapidly, while that into proteins of nucleolar 60-S particles took the maximal value after 20 min. 5 These results may indicate that most of structural proteins of liver ribosomes are synthesized on cytoplasmic ribosomes, then transferred to the nucleolus where their assembly with ribosomal precursor RNA occurs to form 60-S nucleolar particles. The nucleolar 60-S particles may be the precursor to the large subunit of rat liver ribosomes.

Further studies on ribosome unfolding: The reversible release of 5-S RNA

1. The release of 5-S RNA during unfolding of the 50-S ribosomal subunit of Escherichia coli and its reassociation with the unfolded ribosome has been examined. 2. Exposure of ribosomes to 0.5–1.0 M NH 4Cl, at Mg 2+ concentrations of 0.1 mM or lower, results in a partial unfolding of the nucleoprotein particles; 50-S and 30-S subunits are converted to 40-S (38–42-S) and 25-S (24–26-S) components, respectively. 3. As a result of this unfolding, 85–90 % of the 5-S RNA is released from the 50-S ribosomal subunit together with 4–7 ribosomal proteins. 4. The detachment of both 5-S RNA and ribosomal proteins is reversible. When NH 4Cl-treated ribosomes are refolded by dialysis against 10 mM Mg 2+, the 5-S RNA and proteins released at high salt concentrations reassociate with the ribosomal particles. 5. Reattachment of 5-S RNA to 50-S subunits requires the ribosomal protein fraction released by NH 4Cl. The reassociation is specific for 5-S RNA; little tRNA is bound to the ribosomes under these conditions.

Structural and Functional Aspects of Ribosome Interaction with Endoplasmic Reticulum Membranes

The different biochemical components of endoplasmic reticulum membranes (phospholipids, phosphoproteins and RNA) were investigated as to their ability to interact with the ribosomal protein and RNA. Light‐scattering determinations, viscosity measurements and gel‐filtration techniques were employed. A complex between a fraction of ribosomal protein (dialyzed against urea) and a phosphoprotein fraction from endoplasmic reticulum membranes, and a complex between a fraction of ribosomal protein (dialyzed against water) and two different phosphoprotein fractions from endoplasmic reticulum membranes, were obtained. No complexes could be detected between the other components under investigation. Likewise no complexes could be detected in the control experiments with lecithin, pepsin and cytochrome c, tested with ribosomal protein fractions and endoplasmic reticulum membranes phosphoproteins. These results suggest an additional new mechanism concerning the interaction of ribosomes with the endoplasmic reticulum membranes.

Involvement of sulfhydryl groups in the binding of tRNA to reticulocyte ribosomes

The ability of purified reticulocyte ribosomes to bind phenylalanyl-RNA under the direction of polyuridylic acid is inhibited by a number of reagents reactive toward sulfhydryl groups; vicinal dithiols do not seem to be involved. This inhibition occurs in enzymic as well as non-enzymic binding and is also observed in the analogous lysyl-RNA polyadenylic acid system. N-Ethylmaleimide does not inactivate the bindin genzyme, nor does it react with ribosomal RNA. Instead, the site of inhibition is the ribosomal protein fraction. Also, sulfhydryl reagents do not seem to impair the ability of ribosomes to bind polyuridylic acid.

Properties of ribosomal proteins from two mammalian sources

1. Ribosomal protein fractions from rabbit reticulocytes and rat liver were prepared by extracting ribosomes with 0.2n-hydrochloric acid or guanidinium chloride and subsequent dialysis. 2. Treatment for 2.5hr. or less with 0.2n-hydrochloric acid dissolved 46-54% of the proteins, which were richer in arginine and lysine and in N-terminal alanine groups and poorer in aspartic acid and glutamic acid and in N-terminal glycine groups than the acid-insoluble proteins. 3. Protein fractions prepared from the guanidinium chloride extract of ribosomes from rat liver were usually more basic than those from rabbit reticulocytes. 4. The ratios lysine: arginine of fractions in the guanidinium chloride extracts were appreciably higher for proteins from rabbit reticulocytes than from rat liver. 5. The concentration of urea and the pH of the gel affected the rate of migration and number of bands in starch-gel electrophoresis.