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.     

Zinc finger-like motifs in rat ribosomal proteins S27 and S29.

The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins. Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339. Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length. Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541. There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length. Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins. S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes. Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well.     

The complete amino acid sequences of ribosomal proteins L17, L27, and S9 from Bacillus stearothermophilus

The complete primary structures of proteins L17, L27 and S9 extracted from the Bacillus stearothermophilus ribosomes with 1 M NaCl and purified to homogeneity by column chromatography have been determined. The amino acid sequences of these proteins are compared to those of the homologous ribosomal proteins from Escherichia coli. The number of identical amino acid residues between the homologous proteins lies between 33-55%.     

Yeast ribosomal proteins: XII. YS11 of Saccharomyces cerevisiae is a homologue to E. coli S4 according to the gene analysis.

We isolated and sequenced a gene, YS11A, encoding ribosomal protein YS11 of Saccharomyces cerevisiae. YS11A is one of two functional copies of the YS11 gene, located on chromosome XVI and transcribed in a lower amount than the other copy which is located on chromosome II. The disruption of YS11A has no effect on the growth of yeast. The 5'-flanking region contains a similar sequence to consensus UASrpg and the T-rich region. The open reading frame is interrupted with an intron located near the 5'-end. The predicted amino acid sequence reveals that yeast YS11 is a homologue to E. coli S4, one of the ram proteins, three chloroplast S4s and others out of the ribosomal protein sequences currently available.     

A comparative study on 40S ribosomal proteins of Artemia salina and rat liver: micro analysis of amino acid composition by high-performance liquid chromatography

The amino acid compositions of 24 proteins of 40S ribosomal subunits of Artemia salina cysts were determined and compared with those of rat liver. The basic proteins of A. salina 40S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N,N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. salina and rat liver 40S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. salina and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, S16, S17, and S18, strongly related and S14, S15, S20, S23, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui Comparison of the ribosomal protein S11 family

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45–49%) than to the eubacterial counterparts (35%)     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Characterization and primary structure of proteins L28, L33 and L34 from Bacillus stearothermophilus ribosomes.

The complete amino acid sequences of 3 proteins from the 50S subunit of Bacillus stearothermophilus ribosomes were determined by N-terminal sequence analysis and by sequencing of overlapping fragments obtained from enzymatic digestions and chemical cleavages. The proteins BstL28, BstL33 and BstL34, named according to the equivalent proteins in Escherichia coli ribosomes, consist of 60, 49, and 44 amino acid residues and have calculated molecular masses of 6811.0, 5908.6, and 5253.9 Da, respectively. They are highly basic with a content of positively charged residues ranging between 29% for L33 and 45% for L34. The 3 proteins were positioned in the 2-dimensional map of B stearothermophilus 50S ribosomal proteins. The electrophoretic mobilities confirm sizes and net charges deduced from the sequences.     

Yeast ribosomal proteins: XIII. Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7.

We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae. It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI. The haploid strains carrying disrupted YL8A grew more slowly than the parent strain. The open reading frame is interrupted with two introns. The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others.     

The primary structure of rat ribosomal protein L8.

The amino acid sequence of the rat 60S ribosomal subunit protein L8 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L8 has 257 amino acids and has a molecular weight of 28,007. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 4 or 5 copies of the L8 gene. The mRNA for the protein is about 950 nucleotides in length. Rat L8 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.     

The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present

Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.     

Yeast ribosomal proteins: V. Correlation of several nomenclatures and proposal of a standard nomenclature

Summary A tentative nomenclature (YP number) for yeast (Saccharomyces cerevisiae) cytoplasmic ribosomal proteins, which is used in our laboratory (Otaka and Kobata 1978; Higo and Otaka 1979), has been correlated with those of Warner and Gorenstein (1978) and several others. Our nomenclature is based on the two-dimensional gel electrophoretic pattern of proteins as analyzed by a modified method of Mets and Bogorad (1974), while others have used various modifications of Kaltschmidt and Wittmann's two-dimensional gel electrophoresis (1970). The method of correlation involved the examination in our twodimensional electrophoresis system of each protein spot excised from gel patterns prepared by Kaltschmidt and Wittmann's method or vice versa.The numbers of protein species recognized in this paper are 29 for small subunit, and 44 for large subunit. Based on these results, we propose a standard nomenclature for yeast ribosomal proteins, in which the designations YS1–YS29 and YL1–YL44 have been given to the small subunit proteins and the large subunit proteins respectively.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column.     

Purification and characterization of 30S ribosomal proteins from Bacillus subtilis: correlation to Escherichia coli 30S proteins

Summary Twenty proteins were isolated from the 30S ribosomal subunits of Bacillus subtilis and their amino acid compositions and amino-terminal amino acid sequences were determined. These results were compared with the data of Escherichia coli 30S ribosomal proteins and the structural correspondence of individual ribosomal proteins has been established between B. subtilis and E. coli.Post-translational modifications of amino-terminal amino acids of the ribosomal proteins which have been found in E. coli are almost absent in B. subtilis with the exception of acetylated forms of S9.     

The primary structure of rat ribosomal protein L3.

The amino acid sequence of the rat 60S ribosomal subunit protein L3 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L3 has 403 amino acids and has a molecular weight of 46,106. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 9 copies of the L3 gene. The mRNA for the protein is about 1,400 nucleotides in length. Rat L3 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.     

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.