Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
The P-site tRNA Reaches the P/E Position Through Intermediate Positions
While it has been widely accepted that the tRNAs are in A/P and P/E hybrid positions before translocation to the P/P and E/E sites, a recent sm-FRET study suggested that, prior to the binding of EF-G, the ribosome oscillates between three states characterized by three configurations of the tRNAs: (i) the classical state (A/A and P/P), (ii) the hybrid state (A/P and P/E), and (iii) a previously unidentified hybrid state (A/A and P/E), in which the A- and P-site tRNAs have moved independently (Munro et al., Mol. Cell 2007). Here, using cryo-EM and single-particle reconstruction, we studied a pre-translocational ribosome complex that carries a point mutation on the P-loop (G2252C). This complex is known to favor the A/A-P/E hybrid state (Dorner et al., NSMB 2006; Munro et al., Mol. Cell 2007). By employing classification, we obtained several distinct structures of the complex, which confirms the existence of an additional hybrid state of the ribosome (A/A and P/E) suggested by the sm-FRET study. In addition, we have now discovered a transitional position of the tRNA, in which the A-site tRNA remains in its A/A configuration, while the acceptor arm of the P-site tRNA has flipped to make contact with the L1 stalk (Figure 1). Based on these findings, we propose that tRNA moves from the P/P to the P/E hybrid site though intermediate positions, and that the movement is coupled with the ratchet motion of the ribosome: after the peptidyl-transfer reaction, the P-site tRNA apparently oscillates between the classical and the ?flipped? position. As the ribosome starts to ratchet, the L1 stalk moves in toward the inter-subunit space and interacts with the acceptor arm of the P-site tRNA, which temporarily stabilizes the flipped position. Only when the ribosome reaches the fully ratcheted conformation, the tRNA moves from the flipped position to the P/E hybrid site. Our preliminary study on a wild-type pre-transloctional ribosome, in which the hybrid state was stabilized by antibiotic viomycin, also shows the existence of the flipped position. We believe that the intermediate states can be observed since both the point mutation and viomycin slows down the progress of the tRNA through the ribosome.
Figure 1: The different states of the G2252C complex obtained by single particle reconstruction and unsupervised classification. Upper panels: the cryo-EM maps of the 70S ribosome in different states. Lower panels: the cryo-EM maps of the corresponding 50S subunit and the inter-subunit ligands. (A) The classical state of the ribosome. (B) The first intermediate state. (C) The second intermediate state.
1Department of Biomedical Sciences