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1. An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA · L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. 2. Hybridization experiments, using restriction fragments of cDNA for rabbit β globin mRNA, showed that the attached mRNA fragments were derived from the 5′ portion of globin mRNAs. 3. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5′ portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed. [ABSTRACT FROM AUTHOR]

To investigate the mechanism for the transfer of ribosomal proteins from cytoplasm into nuclei and nucleoli, the uptake of ³H-labelled ribosomal proteins by the isolated rat liver nuclei was investigated in the system containing ATP, GTP, CTP, UTP, and an energy-regenerating system. 1. In the presence of cell sap, the transfer became temperature-dependent. The concentration of ribosomal proteins was also very important for their specific transfer and 10-15 µg of ribosomal proteins/ml were suitable in the presence of 107 nuclei. 2. Removal of one of the four nucleoside triphosphates from the complete system containing cell sap, especially that of CTP or UTP, resulted in a marked decrease of the uptake. A low concentration of actinomycin D inhibited significantly the transfer of ribosomal proteins, while α-amanitin to a less extent. The results indicate that the uptake of ribosomal proteins by liver nuclei is largely dependent on RNA synthesis especially rRNA synthesis. 3. The transfer was enhanced to some extent by ATP alone. Other nucleoside triphosphates were less effective. Non-hydrolyzable ATP analogues, adenosine 5'-[β-γ-methylene]triphosphate and adenosine 5'-[α,β-methylene]-triphosphate were only slightly stimulative. Although ATP enhanced the transfer into the extranucleolar fraction to some extent, the maximal transfer not only into nucleoli but also into the extranucleolar fraction was dependent on the rRNA synthesis. 4. Sedimentation analyses of the nucleolar fraction of rat liver nuclei incubated with [³H]GTP or ³H-labelled ribosomal proteins, showed that small but distinct amounts of the both labeled compounds were incorporated into 60S preribosomal particles although most of them were found in ribonucleoprotein particles below 30S which were previously shown to be degraded products containing larger rRNA precursors [Tsurugi, K., Morita, T., and Ogata, K. (1972) Eur. J. Biochem. 25, 117-128]. [ABSTRACT FROM AUTHOR]

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525–531 (1977)]. The optimum pH was 5.5. 2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5. It was inhibited by thiol reagents, E-64, leupeptin and heavy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones, α-N-Benzoylarginine-β-naphthylamide and benzoylarginine amide were not hydrolyzed. [ABSTRACT FROM AUTHOR]

1. Normal and partially hepatectomized rats were pulse labeled for 15 min with [14C]leucine and [3H]leucine respectively. Both kinds of liver homogenates were mixed and the ribosomal protein fraction was purified from the mixed homogenate by chromatographic procedures. The 3H/14C ratio of this fraction was about three times higher than that of total protein, indicating that the synthesis of ribosomal proteins is selectively increased in regenerating rat liver. 2. The free polysomes from normal or regenerating rat livers were incubated with [3H]leucine in the presence of regenerating rat liver supernatant. Ribosomal proteins were then purified, finally by two-dimensional acrylamide gel electrophoresis. Biosyntheses of most of ribosomal proteins by regenerating rat liver polysomes were about 2.5 to 3.6 times higher than those by normal rat liver polysomes. 3. Using a wheat germ cell-free system, the template activities of poly(A)-containing mRNAs for ribosomal proteins were compared between normal and regenerating rat livers. The template activities at 6, 12, 18, 24 and 48 h after partial hepatectomy were about 1.3, 1.8, 2.4, 1.5 and 1.3 times higher, respectively, than the activity of normal rat liver. The extents of increase in the template activity of poly(A)-containing mRNAs for most of individual ribosomal proteins separated by two-dimensional gel electrophoresis were not so different at 18 h after the operation and the average template activity was 2.8 times higher than that of normal rat liver. The enhanced activity of regenerating rat liver for ribosomal protein synthesis is mainly caused by the increase in the apparent concentrations of mRNAs for ribosomal proteins. [ABSTRACT FROM AUTHOR]

The metabolism of newly synthesized ribosomal proteins in regenerating livers of rats pretreated with a low dose of actinomycin D which selectively inhibits rRNA synthesis, has been' investigated. After 15 rain of labeling most newly synthesized proteins were found in the cell sap of regenerating livers of rats pretreated for 1 h with actinomycin D and disappeared, from this fraction during a subsequent chase in the presence of cycloheximide. However, no proteinase activity for ribosomal proteins was detected in cell sap. After treatment in vivo with E-64, a thiol-proteinase inhibitor which inhibits the ribosomal protein-hydrolysing activities of nuclear and mitochondrial plus lysosomal fractions of rat liver, a fraction of newly synthesized ribosomal proteins was found to accumulate in nuclei of actinomycin-D-pretreated regenerating rat liver. These results may indicate that ribosomal proteins synthesized in the absence of rRNA synthesis accumulated in cell sap, and were then transported into nuclei where they were degraded by a proteinase sensitive to E-64. In the absence of actinomycin D pretreatment smaller amounts of ribosomal proteins accumulated in nuclei of regenerating livers of E-64-treated rats. Furthermore, histones (except histone H1) also accumulated in nuclei of regenerating livers of E-64-treated rats with and without actinomycin D pretreatment. Thus, these two kinds of proteins, especially histones, are synthesized in excess of nascent rRNA or DNA, and are degraded preferentially in nuclei in regenerating rat liver. [ABSTRACT FROM AUTHOR]

The effects of ricin and α-sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N-ethylmaleimide after 80S ribosomes were treated with these toxins. By using a double-labelling technique in which ribosomes were incubated with the toxins and then treated with ³H-labelled or 14C-labelled N-ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment wit α-sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and α-sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with α-sarcin alone. These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and α-sarcin on 28S rRNA. [ABSTRACT FROM AUTHOR]

We constructed cDNA libraries from 8-9-S poly(A)-rich RNA from regenerating rat liver, isolated clones specific for ribosomal protein L27 and determined the nucleotide sequences of the cDNA clones. The longest cDNA consists of 15 base pairs from the 5' leading sequence, the entire coding sequence of 411 base pairs, and the 3' trailing sequence of 59 base pairs including the poly(A) tail. The primary structure of protin L27 was deduced from the sequence of nucleotides. Protein L27 contains 135 amino acids and has a molecular mass of 15 666 Da. The amino-terminal sequence agreed well with the published partial amino acid sequence and the calculated amino acid composition is also consistent with the reported composition determined for the hydrolyzate of L27. [ABSTRACT FROM AUTHOR]

cDNA clones specific for rat ribosomal protein L5, which is the protein moiety of 5S RNA protein particles, were isolated by using a mixture of tetradecamer deoxyribonucleotide probes reflecting the amino acid sequence of the N-terminal portion of L5. A cDNA clone (pRL5-1) lacking the 3′-terminal region of L5 was isolated by hybrid-selected translation and shown to be L5-specific by the identity of the deduced amino acid sequence and that of the N-terminus of protein L5. pRL5-1 was then used to select another clone (pRL5-2) by colony hybridization which contained the entire coding region, 3′-non-coding region and poly(A) addition signal. The nucleotide sequence of L5-specific cDNA was determined by using these two clones. It contained a total of 1069 base pairs: 123 base pairs in the 5′-non-coding region, 894 base pairs in the coding region and 52 base pairs in the 3′-non-coding region. The poly(A) addition signal appeared at the 33rd nucleotide after the termination co- don. The amino acid sequence deduced from this nucleotide sequence consists of 296 amino acids. The homology of amino acid sequence between L5 and other 5S RNA-binding proteins is discussed. [ABSTRACT FROM AUTHOR]

A cDNA clone, specific for rat ribosomal protein L31, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of 25 base pairs from the 5' leading sequence, the entire coding sequence of 378 base pairs and the 3' trailing sequence of 36 base pairs besides the poly(A) tail. The primary structure of protein L31 was deduced from the nucleotide sequence. It consists of 124 amino acids with a relative molecular mass of 14331. The calculated amino acid composition is consistent with the reported composition determined for the hydrolysate of L31. The amino acid sequence showed marked homology with that of yeast ribosomal protein L34. [ABSTRACT FROM AUTHOR]

Complexes containing rat liver SOS ribosomes treated with puromycin and high concentrations of KC1, elongation factor 2 (EF-2) from pig liver, and guanosine 5'[β,y-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-inked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by auto radiography. The following proteins of both large and small subunits were identified: L9, LU, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/ S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33 -L11 and LA33-L12. [ABSTRACT FROM AUTHOR]

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8 - 9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the eDNA was determined. It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence of Xenopus lea vis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA.

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel flitration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 45. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30–40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2–4mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol protcases ever known. [ABSTRACT FROM AUTHOR]

Rat liver 80-S ribosomes, irradiated with 254 nm ultraviolet light for 15-60 mm (0.63-2.5 × 105 quanta/ribosome), were treated with sodium dodecyl sulfate; 18-S and 28-S RNA fractions were isolated by sucrose density-gradient centrifugation. The protein moieties of the 18-S and 28-S rRNA fractions were labeled with 125l After digestion of the rRNA fractions with RNase A and T1, the cross-linked proteins were analyzed by two- dimensional acrylarnide gel electrophoresis and then autoradiography. Cross-linked proteins were examined further by dodecyl sullate/acrylamide gel electrophoresis of individual radioactive spots of proteins and their proteolytic peptides on two-dimensional gels. The results showed that S2 and S15 were cross-linked to 18-S rRNA. and L5, L6 and 18 to 28-S rRNA. Similar results were obtained when isolated 40-S or 60-S subunits were irradiated. [ABSTRACT FROM AUTHOR]

The degradations of structural proteins and RNAs of ribosomes in regenerating rat liver were investigated with the following results. 1. Using a modification of the double isotope technique of Arias et al. (1969), it was found that almost all the structural proteins of both large and small ribosomal subunits, separated by dodecylsulfate or two-dimensional polyacrylamide gel electrophoresis, were apparently degraded at the same rates. 2. Studies on the changes in the specific activities of the RNA and protein moieties of the two ribosomal subunits after administration of [Me-³H]methionine as a common precursor showed that their specific activities decreased in parallel, with the same apparent half-life of about 4.5 days. 3. These results suggest that structural components of rat liver ribosomal subunits are degraded as a unit.