Abstract
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.
Highlights
Binding of translocases, EF1-G1⁄7GTP in prokaryotes and EF21⁄7GTP in eukaryotes, to a specific site of the ribosome causes GTP hydrolysis that drives translocation of peptidyl-tRNA from the A-site to the P-site during protein biosynthesis [1,2,3]
It is presumed that the evolutionary divergence between prokaryotic L8 complex and eukaryotic P complex may parallel the luck of sequence similarity at the regions of elongation factor (EF)-G and EF-2 interacting with their respective pentameric complexes
The L8 complex is detached from the GTPase-associated RNA domain within the ribosome in ethanol/NH4Cl [12, 27], and subsequently the rat P complex is incorporated into the core ribosome
Summary
EF1-G1⁄7GTP in prokaryotes and EF21⁄7GTP in eukaryotes, to a specific site of the ribosome causes GTP hydrolysis that drives translocation of peptidyl-tRNA from the A-site to the P-site during protein biosynthesis [1,2,3]. This binding site is nearly equivalent to that for the Escherichia coli L8 complex in 23 S rRNA, as determined by footprinting [23, 24] In contrast with this similar RNA recognition feature of the stalk protein complexes, amino acid sequence homology of each protein constituent is very low between prokaryotic and eukaryotic counterparts [25, 26]. The L8 complex is detached from the GTPase-associated RNA domain within the ribosome in ethanol/NH4Cl [12, 27], and subsequently the rat P complex is incorporated into the core ribosome This replacement of the stalk protein complex changes the specificity of translocase interaction from prokaryotic EF-G to eukaryotic EF-2. The results provide information about conserved features of rRNA involved in the translocase binding
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