Semi-preparative HPLC purification of ribosomal proteins from Bacillus stearothermophilus and sequence determination of the highly conserved protein S19.
Several proteins from the Bacillus stearothermophilus 30S ribosomal subunit which could not be isolated by conventional open-column chromatography were purified by high-performance liquid chromatography using a semi-preparative reverse-phase C4 column. Protein S19 was purified by this technique and the complete amino acid sequence determined. Protein S19 was fragmented and the peptides isolated in picomole quantities were sequenced by an improved manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC) technique; the presence of five consecutive C-terminal lysines in the S19 sequence was confirmed by gas-phase sequencing and fast-atom-bombardment (FAB) mass spectrometry. Protein S19 is composed of 91 amino acid residues which correspond to a molecular mass of 10,428 Da. 71% of the B. stearothermophilus S19 sequence was found to be identical with the corresponding ribosomal protein from Escherichia coli [Yaguchi and Wittmann (1978), FEBS Lett. 88, 227] and both sequences can be aligned without gaps. Among the known 26 amino acid sequences of the B. stearothermophilus and E. coli ribosome such a high degree of conservation has only been observed for a few proteins, all of which are known to be involved in the protein biosynthesis process. Although a clear function has not yet been assigned to protein S19, its high sequence conservation in these two eubacteria clearly indicates an important role of this protein for the function of the ribosome.
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin.
H
H
Hirano
K
K
Eckart
M
M
Kimura
B
B
Wittmann-Liebold
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification*; D012270:Ribosomes / Q000032:analysis
D010446:Peptide Fragments; D012269:Ribosomal Proteins; C040343:ribosomal protein S19