The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

Amino acid sequence of the ribosomal protein L21 of Escherichia coli.

The primary structure of protein L21 from the 50S subunit of Escherichia coli ribosomes has been completely determined by sequencing the peptides obtained by digestion of L21 with trypsin before and after modification of the arginine residues with 1,2-cyclohexanedione, Staphylococcus aureus protease, thermolysin, and pepsin. Automated Edman degradation using a liquid-phase sequenator was carried out on the intact protein as well as on a fragment arising from cleavage with cyanogen bromide. Protein L21 consists of a single polypeptide chain of 103 amino acids of molecular weight 11 565. An estimation of the secondary structure of protein L21 and a comparison with other E. coli ribosomal protein sequences are presented.     

The amino acid sequence of Escherichia coli cyanase

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.     

Genetic position and amino acid replacements of several mutations in ribosomal protein S5 from Escherichia coli.

Summary The relative genetic position of the following four mutations of ribosomal protein S5 has been determined: spc-13, a mutation to spectinomycin resistance; str ⁱ _N421 and str ⁱ _d1023, mutations suppressing dependence on streptomycin and sup _0–1, a mutation suppressing partially the temperature-sensitive phenotype of an alanyl-tRNA synthetase mutation. The transduction experiments performed indicate that the spc-13 site is located in the S5 cistron proximal to the strA locus, that sup _0–1 maps proximal to the aroE gene and that the str ⁱ _N421 and str ⁱ _d1023 loci are located between these two mutational sites.Proteinchemical analysis of the amino acid replacement in protein S5 of strain N421 (carrying the str ⁱ _N421 allele) has shown that an arginine residue is replaced by leucine which results in the appearance of a trypsin intensitive bond between the tryptic peptides T2 and T16. The same alteration has been previously found by Itoh and Wittmann (1973) in the S5 protein of strain d1023.Determination of the alteration of ribosomal protein S5 of strain 0–1 (sup _0–1 allele) revealed that the C-terminal tryptic peptide is altered. It differs from that of the wild-type protein by the lack of five amino acids and the appearance of a C-terminal glycine residue instead of a lysine residue. This change can be explained by the deletion of eleven nucleotides in the S5 cistron of strain 0–1.The recent determination of the primary structure of ribosomal protein S5 (Wittmann-Liebold and Greuer, 1975) allows the ordering of the S5 alterations employed: The order is spc-13-str ⁱ _d1023 (str ⁱ _N421)-sup _0–1 with the spc-13 amino acid replacement being located at the NH₂-terminal portion of the S5 sequence and the alteration of strain 0–1 at the COOH-terminal end. The proteinchemical results are therefore in full agreement with the genetic data and unambiguously allow the conclusion that the S5 cistron is transcribed counterclock-wise on the Escherichia coli chromosome.     

The DNA sequence of the gene rpsA of Escherichia coli coding for ribosomal protein S1.

The DNA sequence of the gene rpsA as well as of its neighboring regions has been determined using the dideoxyribonucleotide method. It was found that there is an "open-reading-frame" of 350 bp which precedes the gene rpsA. Furthermore, an extensive internal repeats of nucleotide sequence have been found in this gene.     

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.     

The histone-like H protein of Escherichia coli is ribosomal protein S3.

We report the purification of four proteins from Escherichia coli that stimulate or inhibit inter- and/or intramolecular recombination promoted by the yeast plasmid-encoded FLP protein. The proteins are identified as the ribosomal proteins S3 (27 kDa), L2 (26 kDa), S4 (24 kDa), and S5 (16 kDa), on the basis of N-terminal sequence analysis. The S3 protein is found to be identical to H protein, an E. coli histone-like protein that is related to histone H2A immunologically and by virtue of amino acid content. The H protein/S3 identity is based on co-migration on polyacrylamide gels, heat stability, amino acid analysis, and effects on FLP-promoted recombination. These results are relevant to current studies on the structure of the E. coli nucleoid. Since the H protein has previously been found associated with the E. coli nucleoid, the results indicate that either (a) some ribosomal proteins serve a dual function in E. coli, or, more likely, (b) ribosomal proteins can and are being mis-identified as nucleoid constituents.     

Primary sequence of the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.     

Structure of the Escherichia coli S10 ribosomal protein operon.

The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.     

Post-translational modification of Escherichia coli ribosomal protein S6.

Summary Escherichia coli has multiple forms of ribosomal protein S6, differing in number of glutamyl residues at the C-terminal end. Three forms are revealed when crude cell extracts are fractionated by a two-dimensional gel electrophoresis technique. Pulse-chase experiments show that the shortes and most alkaline form of S6 is the first to appear. In about one doubling time this form reaches equilibrium with the two other forms of S6, implicating the existance of an enzyme, which adds glutamic acid residues to S6. We show that the relative levels of these three S6 forms are not affected by the growth rate of the culture.     

The amino acid sequence of thiogalactoside transacetylase of Escherichia coli

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.