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

Determination of DNA and RNA in homogenized cells and tissues by surface fluorometry

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

The sedimentation properties of ribosomes and ribosomal subunits from rat liver have been examined in media containing Mg2+ ions at different concentrations.



Purified ribosomes contain 51.4% RNA and 48.6% protein; the phospholipid content is less than 0.1%.



At 10−3 M Mg2+ the S value of monomeric ribosomes is 80 S. At 10−4 M Mg2+ and in Mg2+-free media there are present in addition many particles having a S value of 55 S or 49 S respectively. We have attempted to determine the nature of these particles. All the proteins which we regard as characteristic of 80 S ribosomes, can be extracted from the 55 S particles. The particles also contain two components of RNA sedimenting at a mass ratio of 2:1 at 33 and 18 S, respectively. By raising the concentration of Mg2+ to 10−3 M the 55 S particles revert to 80 S particles. In the presence of EDTA the 55 S particles are dissociated into particles with values of 48 and 28 S.



The separation of the ribosomes into their subunits can be achieved only when all the ribosomal Mg2+ is removed by equimolar amounts of EDTA. The results of short and long time sucrose gradient centrifugation of ribosomes, which were treated before with EDTA clearly show, that the 80 S ribosomes are in an equilibrium with their subunits which is largely on the side of the dissociation products. Complete separation of the subunits with gradient centrifugation is possible only by continuous displacement of the equilibrium during prolonged centrifugation. The subunits have S values of 49 and 29 S in the presence of EDTA, and contain 45.6% RNA.



Studies on the sedimentation characteristics of mixtures of 32P-labelled small subunits with the unlabelled large subunits or with 80 S particles exclude the possibility that the 55 S particle fraction consists of mixture of the 49 S subunit with dimers of the 29 S subunit. Thus our evidence supports the idea that the 55 S and 80 S particles are interconvertible forms of an intact ribosomal particle.



The results are interpreted as indicating that two processes result from a progressive reduction in the amount of Mg2+ bound to the 80 S ribosomes. Firstly, the structure of the subunits is changed and secondly, at the same time, the association of the subunits is loosened due to a stepwise cleavage of the Mg2+ dependent linkages between the subunits. Both processes change the structure of the ribosome such that its S value is reduced from 80 S to lower values. The 49 S and 29 S subunits become separated when the ribosomal Mg2+ is removed quantitatively.

Strukturveränderungen und Dissoziation von Leberribosomen in Abhängigkeit von der Mg++ ‐Konzentration

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. Welfle

Papers

Strukturveränderungen und Dissoziation von Leberribosomen in Abhängigkeit von der Mg++ ‐Konzentration

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.