Translational GTPases and the energy landscape of the 70S ribosome
Final Report Abstract
Translational GTPases (trGTPases) constitute a fundamental group of translation factors that control and steer the ribosome during all four phases of protein synthesis. The major goal of this sub-project was to analyze at the molecular level how trGTPases interact with the ribosome in various functional state and how the intricate interplay between the factors and the ribosome exploits intrinsic large-scale conformational changes of the ribosome to drive the respective steps of protein synthesis. Our work on complexes of the ribosome with EF-G resulted in a revised model for tRNA translocation. It establishes the ribosome as a Brownian ratchet machine that exploits ribosomal intersubunit rotation/back-rotation motions and 30S head swiveling to translocate the tRNAs via novel chimeric intra-subunit hybrid states. Furthermore, a high-resolution structural snapshot of EF-G stabilized in the transition state of GTP hydrolysis on the 70S ribosome reveals how intersubunit rotation of the ribosome is coupled to activation of GTP-hydrolysis. Our structural work on IF2/eIF5B shows reveals a mechanism how the factor exploits rotational motions of the ribosome to load the initiator tRNA and to trigger GTP hydrolysis and thus facilitates ribosomal subunit joining by re-orientating the initiator tRNA. Our work on tRNA selection by EF-Tu supports a seesaw-like model that explains how codon recognition is signaled to the GTPase center of the factor and gives further insight into proofreading. With Zoya Ignatova (P7) we obtained structural information on the post-mitotic eEF1A2 on the 80S. Furthermore, structural profiling of E. coli polysomes reveals differences in translation elongation between bacteria and higher eukaryotes.
Publications
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Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation. Proc. Natl. Acad. Sci. USA 110, 20964-9 (2013)
Ramrath, D.J., Lancaster, L., Sprink, T., Mielke, T., Loerke, J., Noller, H.F. & Spahn, C.M.T.
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EF-G and EF4: translocation and back-translocation on the bacterial ribosome. Nature Reviews Microbiol., 12, 89-100 (2014)
Yamamoto, H., Qin, Y., Achenbach, J., Li, C.M., Kijek, J., Spahn, C.M.T. & Nierhaus, K.H.
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Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV IRES RNA. Nature Struct. Mol. Biol., 21, 721-7 (2014)
Yamamoto, H., Unbehaun, A., Loerke, J., Behrmann, E., Collier, M., Bürger, J., Mielke, T., & Spahn, C.M.T.
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Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA. EMBO J., 34, 3042-58 (2015)
Yamamoto, H., Collier, M., Loerke, J., Ismer, J., Schmidt, A., Hilal, T., Sprink, T., Yamamoto, K., Mielke, T., Bürger, J., Shaikh, T.R., Dabrowski, M., Hildebrand, P.W., Scheerer, P., & Spahn, C.M.T.
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Structural insights into ribosomal rescue by Dom34 and Hbs1 at near-atomic resolution. Nature Commun. 20, 13521 (2016)
Hilal, T., Yamamoto, H., Loerke, J., Bürger, J., Mielke, T., & Spahn, C.M.T.
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Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association. Science Adv. 2, e1501502 (2016)
Sprink, T., Ramrath, D.J., Yamamoto, H., Yamamoto, K., Loerke, J., Ismer, J., Hildebrand, P.W., Scheerer, P., Bürger, J., Mielke, T., & Spahn, C.M.T.
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Ribosomal Chamber Music: Toward an Understanding of IRES Mechanisms. Trends Biochem. Sci. 42, 655-668 (2017)
Yamamoto, H., Unbehaun, A., & Spahn, C.M.T.
