Takuzi Itoh

Ribosomal proteins of bacterial cells: strain- and species-specificity.

Abstract The 50 s ribosomal protein of Escherichia coli labelled with [ 3 H]lysine, and the 30 s protein labelled with [ 14 C]lysine were simultaneously analysed by carboxymethyl-cellulose column chromatography. The results of this study, together with re-analysis by polyacrylamide gel electrophoresis of chromatographically isolated proteins, revealed that the 50 and 30 s subunits which contain respectively at least 15 and 11 main protein components possess almost no common protein components. In addition to the main components several minor protein constituents were detected in both ribosomal subunits. [ 14 C]Lysine-labelled ribosomal protein of E. coli Q13 was compared with [ 3 H]lysine-labelled ribosomal protein of E. coli B by means of chromatography on a carboxymethyl cellulose column. The elution pattern of the 50 s protein was found to be indistinguishable between these two strains, while the 30 s protein of Q13 contained two components which differ chromatographically from those of strain B, the total number of components being the same in each. The same differences in the 30 s protein were found when strains K12 (W1895) and B were compared. The chromatographic comparison of the labelled ribosomal proteins of E. coli and Salmonella abony indicated that the general elution patterns were very similar to each other. Clear differences were however detected in the elution positions of several components of both the 50 s and the 30 s proteins of these two species. [ 3 H]Lysine-labelled protein from Bacillus subtilis ribosomes was compared chromatographically with [ 14 C]lysine-labelled protein from B. megaterium and B. cereus ribosomes to examine the relatedness of species in the genus Bacillus . The results reveal almost no apparent similarities of both the 50 and 30 s proteins among these species. The same situation was found in comparisons of E. coli ribosomal protein with that from several Bacillus species and Sarcina lutea .

Biosynthesis of 50 s ribosomal subunit in Escherichia coli

Abstract Two intermediate particles of 50 s ribosome formation, i.e. [30 s]- and 40 s nascent or precursor particles, were isolated from exponentially growing Escherichia coli cells. Particles indistinguishable from these nascent ribosomal particles were found in the extracts of the cells treated with a low concentration of chloramphenicol. The [30 s]-particles contain undermethylated 23 s rRNA and three main protein components. The 40 s particles also possess undermethylated 23 s rRNA and, in addition to the three protein components found in the [30 s]-particles, 9 other components out of 19 components detectable in the 50 s ribosomal subunit. Both particles include little 5 s RNA. Based on the above findings, a scheme for the biosynthesis of 50 s ribosomal subunits is presented. The protein compositions and 5 s RNA contents of the nascent ribosomal particles are compared with those of protein-deficient particles obtained by exposing 50 s ribosomal subunits to a concentrated LiCl solution. The molecular compositions of these two kinds of particles are found to be similar, but not exactly the same.

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

SummaryThe 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 counter-parts 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.

Primary structure of yeast acidic ribosomal protein YP A1

The ribosomal acidic protein L7/L12 ofEscherichia coli is important in the expression of the function of elongation factors [l]. Ribosomes from other bacterial species have proteins homologous to L7/L12. The ubiquity of these proteins as ribosomal constituents is indicated by a high degree of conservation in their N-terminal amino acid sequence in prokaryotic or in eukaryotic organisms [2--S]. Comparative primary structural analyses of these proteins would give some insights into the evolutionary changes of the ribosomes as well as the nature of the active sites within the molecules. The complete primary structure of an L7/L12equivalent from Bacillus subtilis and its comparison with those from other prokaryotes has been made [6,7]. The primary sequence of an acidic protein from the large subunit of a brine shrimp Artemia salina has been reported and its comparison with other sources presented [8]. We now report the complete primary structure of yeast acidic ribosomal protein, YP Al. This structure is compared with the published primary structures from other organisms such as E. coli [9], B, subtilis [6], Halobacterium cutirubrum [lo] and A. salina [8]. The evolution and correlation of these acidic proteins are discussed.

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf‐SRP54) and the N‐terminal G‐domain and C‐terminal M‐domain (pf‐SRP54M) of pf‐SRP54 with an amino‐terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non‐conserved 213‐nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107‐nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine‐tagged pf‐SRP54M bound specifically to the conserved 107‐nucleotide RNA in the absence of pf‐SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Primary structure of an acidic ribosomal protein from Micrococcus lysodeikticus.

The acidic ribosomal protein L7jLI 2 af~s~~ze~jc~~~ co& is important in the function of elongation factors [l]. Ribosomes from other bacterial species have proteins homologous to L7/L12. The ubiquity of this protein as a ribosomal constituent is indicated by a high degree of cons~Nation in their N-terminal amino acid sequences in prokaryotic or in eukaryotic organisms [2-G]. ~or~~parat~ve primary structural analyses of these proteins would give some insights into the evohrtionary changes of this protein as well as the nature of active sites within the molecule. The complete primary structure of the L7/L12equivak?nt protein, BL9, frorn~aci~~~s s~~bti~i~ has been determined [S] and its comparison with L12 from E. culi shows 50% sequence similarity; position 76-84 (Thr-Gly-Leu-Gly-Leu-Lys-Glu-Ala-Lys), which is a region having predictable &turn or bend structure, is especially highly conserved within these molecules [6]. ~~c~~e~e~~~ ~~~~~~~~~~~~s is a Gram-positive bacterium having the highest ‘X-content of DNA among all the living organisms (72%). We now report the complete primary structure of an a&die ribosomal protein from this ba~te~um, which migrates in the twodimensional gel to approximately the same position as that of E. coli L7/L12 or I

Complete amino acid sequence of an L7/L12-type ribosomal protein from Rhodopseudomonas spheroides☆

. s,ubtililis BL9. The ammo acid sequence of this protein has been compared with the published primary structures of 212_equivalent proteins from other organisms such as & cdi f?],

The primary structure of an acidic ribosomal protein from streptomyces griseus

Abstract The amino acid sequences of an L7/L12-type ribosomal protein (RA) from a photosynthetic bacterium, Rhodopseudomonas spheroides , was determined by manual sequencing of tyrptic, staphylococcal protease and chymotryptic peptides of the protein. The sequence of the amino-termina 50 residues was confirmed by automated Edman degradation of the intact protein in a sequenator. When compared with 1.7/L12-type proteins from other bacteria, the carboxy-termina half of the molecule is highly conserved. The amino-terminal region, however, appears to be less conserved. The presence of tyrosine residue in this protein is the first to be reported for an L7/L12-type acidic ribosomal protein from eubacteria.

Ribosomal Proteins from Streptomycin-resistant and Dependent Mutants, and Revertants from Streptomycin-dependence to Independence in Bacillus subtilis

Abstract The complete primary structure of a L7/L12-type acidic ribosomal protein (SA1) from actinomycetes or mycelial bacterium Streptomyces griseus has been determined. SA1 is composed of 125 amino acid residues and has the composition: Asp 7 , Thr 5 , Ser 5 , Glu 18 , Gln 4 , Pro 4 , Gly 8 , Ala 24 , Val 13 , Met 1 Ile 4 , Leu 12 , Phe 5 , Lys 14 and Arg 1 The molecular weight of SA1 is 13069. The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a Beckman sequenator, and 4- N , N -dimethylaminoazobenzene-4′-isothiocyanate/phenylisothiocyanate double-coupling degradafion of the peptides obtained by digestions with trypsin, chymotrypsin, pepsin and Staphylococcus aureus protease of the intact protein. When the amino-terminal sequence of the SA1 (the first 39 residues) was compared to those of equivalent proteins from other eubacteria, the highest degree of similarity was found with that from Arthrobacter glacialis . The acidic ribosomal proteins from these two Gram-positive bacteria (S. griseus and A. glacialis) are somewhat distinct, in amino acid sequence and in amino acid compositions, from those of other Gram-negative and Gram-positive bacteria so far studied. However, they still appear to retain the characteristic prokaryotic-type structure.

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freundii

SummaryStreptomycin-resistant and dependent mutants were isolated from Bacillus subtilis ATCC 6633. Ribosomal proteins were analyzed from six such mutants with chromatography on carboxymethyl cellulose or phosphocellulose columns. An altered specific 30s ribosomal protein, 30B, could be detected in all of these mutants. Streptomycin-independent revertants were isolated from a streptomycin-dependent strain. Some of them had an altered 30s ribosomal protein, 30A, and some others had an altered 30C protein. It was shown that from the data of partial N-terminal amino acid sequences together with amino acid compositions and mobilities on two-dimensional gel electrophoresis of these proteins that 30A, 30B and 30C proteins were homologous with S5, S12 and S4 of E. coli 30s ribosomal proteins, respectively.