Book of Abstracts: Albany 2007
June 19-23 2007
The transfer RNA in the Hybrid P/E State
In the course of the elongation cycle, tRNA traverses through the ribosome, transiently appearing in the hybrid P/E state, characterized the anticodon paired with the codon at the P site of the small subunit, and the deacylated CCA end bound at the E site of the large subunit [1,2]. A previous cryo-EM map of the ribosome in this translocating state showed that the CCA end is rotated around the anticodon stem loop by about 35º, and the elbow is notably displaced from its position in the P/P site .
In the current study we have correlated the cryo-EM density of the P/E-state tRNA with snapshots along trajectories (longest run for 45 ns) of the unbound tRNA molecule, obtained by molecular dynamics simulations (see similar correlative study for GTPase-associated center ). The best-matching atomic structures, when placed into the context of the x-ray structure of the ribosome, show how the tRNA can adopt the observed conformation as part of its dynamic spectrum of conformations and how it might interact with the ribosome. The results include the following observations: (i) identification of low-density regions in the cryo-EM map, providing an indication for the locations of unstable elements in the tRNA; (ii) a sharply twisted CCA end, a kinked and twisted anticodon stem, and a highly mobile elbow, which are among the features distinct for the P/E-state tRNA, as opposed to its L-shaped appearance in any other observed states; (iii) the P/E-state tRNA interacts with the ribosome at both P and the E sites in a way that is different from its interactions in the P/P and E/E positions. Specifically, at the CCA end, helix 68 is seen to shift toward the L1 lobe and interacts with nucleotides in tRNA around positions 5 and 69. At the anticodon end, the A790 loop is out of the tRNA?s way moving toward the E site due to the kinked conformation in the anticodon stem; (4) the key process in translocating tRNA from the P site to the P/E site is the release of the tRNA from binding with helix 69, inducing a cascade of conformational changes, propagating from H68 to the L1 lobe.
This study has not only provided insights into conformation and ribosome interactions of the P/E-state tRNA, but has also demonstrated a new way to analyze cryo-EM maps in terms of the dynamical features of the molecule visualized.
References and Footnotes
Wen Li1 and Joachim Frank2, 3