Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding
T. Uchiumi et al., Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding, J BIOL CHEM, 274(39), 1999, pp. 27578-27582
The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli rib
osomes 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 MgCI2, 0.5 M
NH4Cl, 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 fragm
ent 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 th
is incorporation. The resultant hybrid ribosomes showed eukaryotic transloc
ase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent,
GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent act
ivity was dependent upon the P complex binding and was inhibited by the ant
ibiotic thiostrepton, a ligand for a portion of the GTPase-associated domai
n of prokaryotic ribosomes, This hybrid system clearly shows significance o
f binding of the P complex to the GTPase-associated RNA domain for interact
ion of EF-2 with the ribosome. The results also suggest that E, coli 23 S r
RNA participates in the eukaryotic translocase-dependent GTPase activity in
the hybrid system.