Rat Liver Ribosomal Proteins Research Articles

Identification of human, rat and chicken ribosomal proteins by a combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

To identify the exact spot position of human, rat and chicken ribosomal proteins (RP) separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), a 2-DE system was designed to separate RP with a pI>8.6 according to their charge in the first dimension and to their molecular mass in the second dimension. Individual proteins were excised from the gels and identified by mass spectrometry after digestion by trypsin. In addition, a mixture of purified RP from these three species was also analyzed by tandem mass tag spectrometry. By combining those two methods 74 RP from human, 76 from rat and 67 from chicken were identified according to the nomenclature initially defined for rat liver RP and by using the Swiss-Prot/trEMBL databases. Whereas human and rat RP were well described, most of RP from chicken were not characterized in databases, since 35 out of 67 chicken RP identified in this study were not listed yet. We propose here the first comprehensive description of chicken RP and their comparison to those from human and rat.

Crystallization and preliminary X-ray study of an N-terminal fragment of rat liver ribosomal P2 protein.

Ribosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)(2)-P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1-65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4 A resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0 A.

Role of 5SrRNA as a Positive Effector of Some Aminoacyl-tRNA Synthetases in Macromolecular Complexes, with Specific Reference to Methionyl-tRNA Synthetase

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization and nucleotide sequence of a transcriptional unit ofMethanococcus vannielii comprising genes for protein synthesis elongation factors and ribosomal proteins

By a chromosome walking strategy the DNA region from Methanococcus vannielii flanking the genes for protein synthesis elongation factor (EF) 1 alpha and EF-2 was cloned and sequenced. A gene organization of 5' - beta' - open reading frame (ORF) 1 - ORF2 - S12 - S7 - EF-2 - EF-1 alpha - S10 - ORF3 - ORF4 - 3' was found where beta', S12, S7, S10, EF-2, and EF-1 alpha represent gene products with sequences similar to the beta' subunit of RNA polymerase, ribosomal proteins S12, S7, and S10, and EF-G and EF-Tu from Escherichia coli, respectively. ORF1-4 represent gene products with no known eubacterial counterparts. Northern blot analysis of transcripts and nuclease S1 mapping showed that transcription initiates between beta' and ORF1 and terminates at the 3' side of the S10 gene and that the genes from ORF1 to S10 are cotranscribed. Apart from the presence of two additional ORFs, ORF1 and ORF2, and of the gene for S10, this organization is identical to that of the eubacterial "streptomycin operon." ORF1 displays sequence similarity to rat liver ribosomal protein L30 and may represent one of the "additional" ribosomal proteins of Methanococcus. The sequenced part of the beta' gene and the EF-2 and EF-1 alpha gene products from Methanococcus are more similar to their eukaryotic than to their eubacterial counterparts. It appears, therefore, that the genetic organization of the translational components resembles the situation in eubacteria, whereas their primary structures are more eukaryotic in nature.

A comparison of accessibility of ribosomal proteins on free and membrane-bound ribosomes: The ribosomal proteins potentially involved in ribosome-membrane binding

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using membrane-bound and free ribosomes. Comparisons indicated that 12–13 large ribosomal proteins are masked by ribosomal association with membranes. These consisted of L8, L10, L17, L26–28, L31 and L36, and probably also include L4, L5, L7 and L29. These proteins seem to surround a region centered about L3 and may partly define a ribosomal channel through which the nascent peptide emerges. Approx. 10–20% of the large ribosomal subunit surface area is shielded by the membrane.

The primary structures of ribosomal proteins S14 and S16 from the archaebacterium Halobacterium marismortui. Comparison with eubacterial and eukaryotic ribosomal proteins.

The amino acid sequences of two ribosomal proteins, S14 and S16, from the archaebacterium Halobacterium marismortui have been determined. Sequence data were obtained by the manual and solid-phase sequencing of peptides derived from enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease as well as by chemical cleavage with cyanogen bromide. Proteins S14 and S16 contain 109 and 126 amino acid residues and have Mr values of 11,964 and 13,515, respectively. Comparison of the sequences with those of ribosomal proteins from other organisms demonstrates that S14 has a significant homology with the rat liver ribosomal protein S11 (36% identity) as well as with the Escherichia coli ribosomal protein S17 (37%), and that S16 is related to the yeast ribosomal protein YS22 (40%) and proteins S8 from E. coli (28%) and Bacillus stearothermophilus (30%). A comparison of the amino acid residues in the homologous regions of halophilic and nonhalophilic ribosomal proteins reveals that halophilic proteins have more glutamic acids, asparatic acids, prolines, and alanines, and less lysines, arginines, and isoleucines than their nonhalophilic counterparts. These amino acid substitutions probably contribute to the structural stability of halophilic ribosomal proteins.

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca 2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 × 10 4. When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 × 10 4 was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca 2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A) + RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S 17 protein (pRS 17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid(aa) sequence of S 17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an M r of 15377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an M r of 12652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3 ' -noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].

Yeast ribosomal proteins

Two proteins, YL41 and YL43, were isolated from 80S ribosomes of Saccharomyces cerevisiae by filtration through a Sephacryl S-200 column and by chromatography on a column of carboxymethylcellulose. Their amino acid compositions are presented. Twenty-four proteins including these two proteins were subjected to sequence analyses by automated Edman degradation. Amino-terminal amino acid sequences were determined for 17 proteins,YS3, YS9, YS23, YS24, YS29, YL6, YL8, YLll, YLI5,YL17, YL23, YL28, YL33, YL37, YL39, YL41, and YL43.YL41, which has a 72.7% lysine and arginine content, was found to be particular to eukaryotic ribosomes. The amino-termini of another seven proteins, YS2, YS5, YS8, YS12,YS13, YS20, and YS27, were suggested to be blocked. Comparison of the amino-terminal sequences with all other ribosomal protein sequences so far available indicates that YS9 shows sequence homology to rat liver ribosomal protein S8 (Wittmann-Liebold et al. 1979).

The primary structure of rat liver ribosomal protein L39.

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.

Ribosome conformational changes associated with protein S6 phosphorylation.

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using ribosomes with unphosphorylated, and extensively phosphorylated S6. Comparison of the results indicated that proteins S3, S4, S7, and S23/24 of the small subunit, and proteins L9, L10, L12, L18, L27, L34, and L36 are involved in a ribosomal conformational change.

Affinity labelling of rat liver ribosomal protein S26 by heptauridylate containing a 5'-terminal alkylating group.

Heptauridylate bearing a radioactive alkylating [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to the 5'-phosphate via amide bond, was bound to ribosomes and small ribosomal subunits from rat liver which thereby were coded to bind N-acylated Phe tRNA. After completion of the alkylating reaction and subsequent hydrolysis of the phosphamide bond ribosomal proteins were isolated. Radioactivity was found covalently associated preferentially with protein S26 and, to a very small extent, with proteins S3 and S3a. The affinity labelling reaction could be abolished by (pU)14 and poly(U). From the results it is concluded that ribosomal protein S26 is located at the mRNA binding site of rat liver ribosomes.

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Yeast ribosomal proteins: VII. Cytoplasmic ribosomal proteins from Schizosaccharomyces pombe

The cytoplasmic ribosomal proteins from a fission yeast Schizosaccharomyces pombe were analysed by two-dimensional polyacrylamide gel electrophoresis. Seventy-three protein species were identified in the 80S ribosome, and named SP-S1 to SP-S33 and SP-L1 to SP-L40 in the small and large subunits, respectively. Many of these proteins could be correlated to those of Saccharomyces cerevisiae on the basis of their electrophoretic mobilities. Eleven proteins were isolated from the 80S ribosome, and their amino acid compositions were determined. Of these, SP-S6, SP-L1, SP-L12, SP-L15, SP-L17, SP-L27, SP-L36 and SP-L40c and d were sequenced from their amino-termini. SP-S28 and SP-L2 appear to have their amino-termini blocked. These results were compared with the data available for the S. cerevisiae and rat liver ribosomal proteins. The S. cerevisiae counterparts of the eight proteins mentioned above were found to be YS4, YL1, YL10, YL14, YL35, YL40 and YL44c and d, respectively. The rat liver counterparts of SP-S6, SP-L1, SP-L27 and SP-L40c and d were the rat S6, L4, L37 and P2, respectively. Comparison of the partial sequences of these ribosomal proteins suggests that these two yeasts are relatively far apart, phylogenetically.

Separate binding sites on rat liver ribosomal protein L6 for 5 S and 5.8 S ribosomal ribonucleic acids and for transfer ribonucleic acids.

The rat liver ribosomal protein L6 binds to 5 S and 5.8 S rRNAs, and to initiator and elongator tRNAs. Experiments were carried out to determine if the protein has separate domains for binding each of these nucleic acids. For that purpose, nucleic acid.L6 complexes were immobilized on Sepharose and their capacity to retain 32P-labeled nucleic acids was assessed. A 5 S rRNA.L6 affinity complex binds 5.8 S [32P]rRNA indicating that L6 has separate binding sites for 5 S and 5.8 S rRNAs. A 5 S rRNA.L6 affinity complex also binds [32P]tRNAPhe, and unlabeled 5.8 S rRNA does not compete with the radioactive tRNA for binding to L6, suggesting that the ribosomal protein has a third, distinct, nucleic acid-binding domain. To determine if L6 has separate sites for the binding of elongator and initiator tRNAs, tRNAPhe. L6 and tRNAfMet.L6 affinity columns were constructed. The tRNAPhe.L6 affinity complex binds [32P]tRNAfMet, and the tRNAfMet.L6 complex binds [32P]tRNAPhe, suggesting there are distinct sites on L6 for the interaction with initiator and elongator tRNAs; however, competition experiments imply that, while there are two sites for binding tRNAs to L6, the sites do not discriminate between initiator and elongator tRNAs.

The primary structure of the acidic phosphoprotein P2 from rat liver 60 S ribosomal subunits. Comparison with ribosomal 'A' proteins from other species.

The primary structure of rat liver ribosomal protein P2 was deduced from the sequence of the peptides. Ten peptides were obtained by cleavage of P2 with trypsin. The peptides, which accounted for the 111 residues of P2, were isolated by high voltage electrophoresis and chromatography on cellulose thin layer sheets, and the partial or complete sequence was determined by micromanual or solid-phase procedures using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate and phenylisothiocyanate. In a similar manner, the sequence of 14 peptic peptides was determined. The sequence of the NH2-terminal 30 residues of P2 was obtained by automatic Edman degradation in a sequenator. The ordering of the tryptic peptides was aided by determination of the partial or complete sequence of fragments generated with chymotrypsin, or Armillaria mellea protease, or by secondary cleavage of peptic peptides with trypsin. The carboxyl-terminal sequence was obtained from a cyanogen bromide fragment and from hydrolysis with carboxypeptidase. The sequence of protein P3 was also determined. P3 differs from P2 only in that it lacks the carboxyl-terminal 8 residues, and hence, it is likely to be a proteolytic product of P2. Rat liver ribosomal protein P2 is homologous with yeast YP A1, with Artemia salina eL12, and with Halobacterium cutirubrum L20. It is likely that rat liver P2 is also homologous with the prokaryotic ribosomal "A" proteins, Escherichia coli L7/L12, Micrococcus lysodeikticus MA1, and Bacillus subtilis L9, but that during evolution, a transposition of a portion of the molecule occurred.

Isolation and characterisation of cyclic AMP-dependent phosphorylation sites from rat liver ribosomal protein S6

Isolation and characterisation of cyclic AMP-dependent phosphorylation sites from rat liver ribosomal protein S6

Alterations in DNA-dependent RNA polymerase I from rat liver accompanying ethionine administration

Multiple injections of ethionine plus adenine resulted in 3- to 4-fold increases in the activity of RNA polymerase I from rat liver nuclei, whereas the activity of RNA polymerase II was relatively unaffected. Methyl-deficient preribosomal RNA was present in the livers of rats after treatment for 2 days. Both incorporation of labelled orotate into rat liver ribosomal RNA and protein synthesis in polysomes gradually increased.