Determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.
Limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. Surface sites of 14 ribosomal proteins from Escherichia coli 50S subunits were determined using proteases with different specificities. To assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from Bacillus stearothermophilus were also subjected to limited proteolysis. The results obtained indicate a conservation of the three-dimensional ribosomal structure at the peptide level. The data for the eubacterial ribosomes are in full agreement with the model of the 50S protein topography derived from immunological data. Furthermore, peptide surface regions of archaebacterial ribosomes have been investigated. The results presented in this work prove that limited proteolysis can successfully be applied to halophilic and thermophilic ribosomes from archaebacteria.
Kruft
V
V
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Federal Republic of Germany.
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D005075:Biological Evolution; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000235:genetics; D006217:Halobacterium / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure; D013379:Substrate Specificity; D016944:Sulfolobus / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D012269:Ribosomal Proteins; D010450:Endopeptidases