The primary structure of ribosomal protein S7 from E. coli strains K and B.

Ribosomal proteins S7 from 30S subunits of Escherichia coli strains K and B differ extensively in their aminoacid compositions. The experimental details which led to the determination of the complete primary structures of proteins S7K and S7B are presented. Protein S7K consists of a single polypeptide chain of 177 aminoacids giving a calculated molecular weight of 19, 732, whereas protein S7B has 153 residues which amount to a molecular weight of 17,131. Aminoacid sequences were determined by a combination of automated Edman degradation of the intact proteins in a modified Beckman sequenator and sequencing of peptides obtained by digestion with trypsin. Staphylococcus aureus protease, thermolysin and pepsin, either by solid-phase Edman degradation or by dansyl-Edman degradation. Additional information about the primary structure was derived from peptides resulting from chemical cleavages of the protein by 2-(2-nitrophenyl-sulphenyl)-3-methyl 3' bromoindolenine at its tryptophanyl bonds and by cyanogen bromide at its methionyl bonds leading to large fragments. The mutational event occurring between S7B and S7K was characterized. Protein S7K contains an additional sequence of 24 aminoacids at its C-terminal end. The aminoacid sequence of both proteins S7K and S7B was compared to the published sequences of the other ribosomal proteins of Escherichia coli and predictions for the secondary structure of these proteins were made.     

The primary structure of rat ribosomal protein S7

The amino acid sequence of the rat 40S ribosomal subunit protein S7 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein. Ribosomal protein S7 has 194 amino acids and has a molecular mass of 22,113. Hybridization of the cDNA to digest of nuclear DNA suggests that there are 14-16 copies of the S7 gene. The mRNA for the protein is about 725 nucleotides in length. Rat S7 is homologous with Xenopus laevis S8. The protein contains a possible internal duplication of 10 residues.     

The functional and structural homology of ribosomal protein S7 of E. coli strains K and MRE600

Summary The 30S ribosomal protein S7 purified from E. coli MRE600 displaces specifically and stoichiometrically the endogenous K-S7 protein when it is added to a reconstitution system containing total K strain 30S protein and 16S RNA. The S7 proteins from the two strains have been shown to contain a group of common trypic peptides and to crossreact immunologically. Therefore, the 30S ribosomal protein S7 from E. coli K strain and MRE600 are functionally and structurally homologous despite differences in amino acid composition, molecular weight and electrophoretic mobility.     

Functional homology between the 30S ribosomal protein S7 from E. coli K12 and S7 from E. coli MRE600

Summary It is known that the 30S protein S7 from E. coli strain MRE600 is chemically different from the S7 from E. coli strain K12 (Q13). We have reconstituted 30S subunits using S7 from MRE600 and all other molecular components from K12 and compared the functional activity of the reconstituted particles with those of the particles reconstituted using the S7 from K12. Both reconstituted particles showed the same activity in several functions tested. Since the presence of S7 is essential for the reconstitution of active 30S subunits, we conclude that the S7 from strain K12 is functionally equivalent to the S7 from strain MRE600.This is paper No. 1612 of the Laboratory of Genetics and paper XVIII in the series, Structure and Function of Bacterial Ribosomes. Papers XVI and XVII in this series are Fahnestock, Held, and Nomura (1972) and Fahnestock, Erdmann, and Nomura (1973), respectively.     

Changes in the primary structure of a mutationally altered ribosomal protein S4 of Escherichia coli

Summary Protein S4-su6 is a mutationally altered 30 S protein from Escherichia coli. S4-su6 suppresses the expression of streptomycin dependence controlled by another 30S protein. In addition, S4-su6 binds less tightly to its RNA binding site than the wild type form of S4. The present data show that at least seven amino acid replacements have occured in S4-su6. Moreover, the tryptic peptide pattern of S4-su6 is different from that of S4. Therefore, the pleiotropic effects of the mutation responsible for the altered structure of S4-su6 can be accounted for by extensive changes in the primary structure of S4-su6.Supported by the Deutsche Forschungsgemeinschaft as well as the University of Uppsala.     

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.     

Sequence determination of protein S9 from the Escherichia coli ribosome.

The complete amino acid sequence of ribosomal protein S9 of Escherichia coli has been established. The protein was digested with trypsin and Staphylococcus aureus protease and the resulting peptides were separated by ion exchange chromatography on a new Dowex 50W-X7 microcolumn or a small phosphocellulose column. If necessary, they were rechromatographed on purified cellulose thin-layer plates on a preparative scale. The sequences of the peptides were determined by the micro dansyl-Edman technique, whereas the alignments of the tryptic peptides were mainly established from large cyanogen bromide fragments which were sequenced by the automatic Edman degradation process. Protein S9 is 128 amino acids long and has the following composition: Asx7, Thr5, Ser7, Glx16, Pro3, Gly13, Ala10, Val10, Met3, Ile7, Leu9, Tyr5, Phe4, His1, Lys10, Arg18. The molecular weight as calculated from the amino acid composition is 14 569. A total of 92.6 mg of the lyophilized protein was used for the determination of the primary structure of S9. Most of the material was needed to isolate sufficient amounts of the CNBr-fragments for the automatic degradation in the sequenator.