Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding.
The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.
Uchiumi
T
T
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan. uchimi@giptc.shinshu-u.ac.jp
Hori
K
K
Nomura
T
T
Hachimori
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013883:Thiostrepton / Q000494:pharmacology
D029968:Escherichia coli Proteins; D020652:Peptide Elongation Factor 2; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; C000630686:RPL10 protein, human; C053634:RPL7 protein, human; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; C016700:ribosomal protein L7-L12; D020558:GTP Phosphohydrolases; D013883:Thiostrepton