Article abstract
Nature Structural & Molecular Biology 15, 910 - 915 (2008)
Published online: 17 August 2008 | doi:10.1038/nsmb.1469
A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation
Sean R Connell1,2,9, Maya Topf3,9, Yan Qin4,8,9, Daniel N Wilson4,5,6, Thorsten Mielke7, Paola Fucini2,4, Knud H Nierhaus4 & Christian M T Spahn1
Abstract
EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.
- Institut für Medizinische Physik und Biophysik, Charite–Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117-Berlin, Germany.
- Cluster of Excellence for Macromolecular Complexes, Institut für Organische Chemie und Chemische Biologie, J.W. Goethe Universität, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany.
- Institute of Structural and Molecular Biology, School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.
- Max Planck Institute für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, 14195-Berlin, Germany.
- Gene Center and Department for Chemistry and Biochemistry, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
- Munich Center for Integrated Protein Science (CiPSM), University of Munich, 81377 Munich, Germany.
- UltraStrukturNetzwerk, Max Planck Institute für Molekulare Genetik, Ihnestrasse 73, 14195-Berlin, Germany.
- Present address: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
- These authors contributed equally to this work.
Correspondence to: Christian M T Spahn1 e-mail: christian.spahn@charite.de
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