The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.
It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.
Stöffler-Meilicke
M
M
Max-Planck-Institut für Molekulare Genetik, Abt Wittmann, Berlin, Dahlem, Germany.
Stöffler
G
G
D001105:Archaea / Q000737:chemistry*; D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D017017:Methanococcus / Q000737:chemistry; D008854:Microscopy, Electron; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure; D016944:Sulfolobus / Q000737:chemistry
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; C016700:ribosomal protein L7-L12