Determination of the number of proteins in liver ribosomes and ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis.

Synthesis of ribosomal proteins takes place in the cytoplasm and is independent of nuclear synthesis of ribosomal RNA.

Persistent cytoplasmic synthesis of ribosomal proteins during the selective inhibition of ribosomal RNA synthesis.

Molecular weight distribution of proteins in rabbit reticulocyte ribosomal subunits.

The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

Divergence exists between eight ribosomal proteins of rat and rabbit. This is the first time such a divergence has been precisely demonstrated among mammals. In addition, a proteolytic activity, giving the appearance of modified proteins, is observed in the liver of both species but not in rabbit reticulocytes. These results were made possible by a recently developed method. Ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis in four different but related systems. The precise position of each individual protein was established in each of the four systems. Certain ribosomal proteins from different tissues, seemingly identical in one system were found to be different in other systems. Significant differences occurred between proteins of each ribosomal subunit from the two different species. Variation between reticulocyte and liver ribosomal proteins of the rabbit were minor. Several liver proteins of both species change position or disappear and apparently new proteins appear, if appropriate steps are not taken to prevent proteolysis. Differences in behavior of three small subunit and five large subunit proteins are attributable to an evolutionary divergence between the two species.

Differences in Electrophoretic Behaviour of Eight Ribosomal Proteins from Rat and Rabbit Tissues and Evidence for Proteolytic Action on Liver Proteins

Total ribosomal protein from rat liver ribosomes can be separated into about 20 chief electrophoretic fractions by preparative polyacrylamide gel electrophoresis. Ten electrophoretically homogeneous fractions have been isolated from the total mixture of ribosomal protein, respectively from proteins, prefractionated by CM-cellulose chromatography. Amino acid composition and molecular weights of some fractions have been determined. The amino acid composition of these fractions and of the total protein mixture are basically similar but there are also significant differences with regard to some amino acids. The molecular weights of the proteins studied are in the range between 7,000 and 29,000.

Studies on proteins of animal ribosomes. II. Separation of ribosomal proteins from rat liver by preparative polyacrylamide gel electrophoresis and some properties of the protein fractions.

H. Bielka

Papers

Studies on proteins of animal ribosomes. II. Separation of ribosomal proteins from rat liver by preparative polyacrylamide gel electrophoresis and some properties of the protein fractions.