Mechanisms regulating speed and accuracy of translation
Final Report Abstract
The major goal of the proposal was to understand the speed and accuracy of translation and its effect on co-translational folding. One of the major achievements is the discovery of the function of EF-P, a specialized factor that facilitates peptide bond formation between consecutive proline residues. In collaboration with Daniel Wilson, Roland Beckmann, Nedijko Budisa, Andrea Vaiana and Helmut Grubmüller we showed that EF-P acts by entropic steering of Pro-tRNA towards a catalytically productive orientation in the peptidyl transferase center of the ribosome. In collaboration with the Reinhard Lipowsky/Sophia Rudorf groups, we developed a stochastic model of translation, which now allows us to model continuous and discontinuous translation regimes, decipher kinetics of cotranslational protein folding, or predict the translational efficiency of a given mRNA. As an additional factor for tuning the translational efficiency, we studied the mechanism of translation initiation in polysomes and show that mRNA with a long 5′UTR can recruit the ribosome to a stand-by site when the genuine start site is still occupied by the preceding ribosome, which results in high density of ribosome packing in the polysome and ensures high translational efficiency of the given mRNA. We also identified an unexpected consequence of ribosome pausing at a codons that are intrinsically slippery, that is are prone to frameshifting: when an aminoacyl-tRNA for one of the slippery site codons is not available, the ribosome switches to an alternative reading frame independent of the presence of the downstream regulatory mRNA structures that stimulate programmed ribosome frameshifting. One of the new directions was to establish real-time approaches to study cotranslational protein folding. One important result was to show that the folding of a small alpha-helical N-terminal domain of HemK, a conserved methyltransferase, takes a different pathway in solution and on the ribosome. While in solution the domain folds rapidly and in a concerted fashion from an unfolded to the native state, its co-translational folding is sequential, reflecting the vectorial nature of protein synthesis, and limited by the rate of translation. Introduction of rare codons at non-native positions in an all-beta protein gamma-B crystallin results not only in slowing down of translation, but also in slower domain folding, leading to an altered spectrum of protein conformations, lower protein solubility and higher susceptibility of the protein to proteases. In collaboration with Holger Stark and Helmut Grubmüller, we solved the mechanism of UGA recoding by selenocysteine and revealed how codon recognition triggers GTP hydrolysis in translational GTPases, which finally solved a major question as to how the fidelity of codon-anticodon interaction regulates the velocity of GTP hydrolysis. Our biochemical work shed light onto the chemical mechanism of GTP hydrolysis by EF-Tu, which is likely conserved between different translational GTPases. Together with Daniel Wilson, we provided new insights into the mechanism of translation termination and in particular in the dynamics of translational termination factors on the ribosome. Finally, we developed approaches to estimate error frequencies of translation in vivo by unbiased, highly accurate mass spectrometry methods. We show that frequencies of misreading errors are highly non-uniform, ranging from <10^-7 up to 10^-3. Most frequent mismatches result from the G-U mismatch in the codon-anticodon complex. Together, our data provide new insights into the mechanisms of translation, translational fidelity and co-translational protein folding.
Publications
- EF-P is essential for rapid synthesis of proteins containing consecutive proline residues. Science 339, 85- 88 (2013)
Doerfel, L.K., Wohlgemuth, I., Kothe, C., Peske, F., Urlaub, H., and Rodnina, M.V.
(See online at https://doi.org/10.1126/science.1229017) - Energy barriers and driving forces in tRNA translocation through the ribosome. Nat Struct Mol Biol 20, 1390-1396 (2013)
Bock, L.V., Blau, C., Schröder, G.F., Davydov, II, Fischer, N., Stark, H., Rodnina, M.V., Vaiana, A.C., and Grubmüller, H.
(See online at https://doi.org/10.1038/nsmb.2690) - Deducing the kinetics of protein synthesis in vivo from the transition rates measured in vitro. PLoS Comput Biol 10, e1003909 (2014)
Rudorf, S., Thommen, M., Rodnina, M.V., and Lipowsky, R.
(See online at https://doi.org/10.1371/journal.pcbi.1003909) - Ribosome-induced tuning of GTP hydrolysis by a translational GTPase. Proc Natl Acad Sci USA 111, 14418-14423 (2014)
Maracci, C., Peske, F., Dannies, E., Pohl, C., and Rodnina, M.V.
(See online at https://doi.org/10.1073/pnas.1412676111) - Cotranslational protein folding on the ribosome monitored in real time. Science 350, 1104-1107 (2015)
Holtkamp, W., Kokic, G., Jäger, M., Mittelstaet, J., Komar, A.A., and Rodnina, M.V.
(See online at https://doi.org/10.1126/science.aad0344) - Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome. J Am Chem Soc 137, 12997-13006 (2015)
Doerfel, L.K., Wohlgemuth, I., Kubyshkin, V., Starosta, A.L., Wilson, D.N., Budisa, N., and Rodnina, M.V.
(See online at https://doi.org/10.1021/jacs.5b07427) - Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling. Nat Commun 7, 11657 (2016)
Katoh, T., Wohlgemuth, I., Nagano, M., Rodnina, M.V., and Suga, H.
(See online at https://doi.org/10.1038/ncomms11657) - The pathway to GTPase activation of elongation factor SelB on the ribosome. Nature 540, 80-85 (2016)
Fischer, N., Neumann, P., Bock, L.V., Maracci, C., Wang, Z., Paleskava, A., Konevega, A.L., Schröder, G.F., Grubmüller, H., Ficner, R., Rodnina, M.V., and Stark, H.
(See online at https://doi.org/10.1038/nature20560) - Structural basis for polyprolinemediated ribosome stalling and rescue by the translation elongation factor EF-P. Mol Cell 68, 515- 527 (2017)
Huter, P., Arenz, S., Bock, L.V., Graf, M., Frister, J.O., Heuer, A., Peil, L., Starosta, A.L., Wohlgemuth, I., Peske, F., Novacek, J., Berninghausen, O., Grubmüller, H., Tenson, T., Beckmann, R., Rodnina, M.V., Vaiana, A.C., and Wilson, D.N.
(See online at https://doi.org/10.1016/j.molcel.2017.10.014) - Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1. Nat Commun 9, 3053 (2018)
Graf, M., Huter, P., Maracci, C., Peterek, M., Rodnina, M.V., and Wilson, D.N.
(See online at https://doi.org/10.1038/s41467-018-05465-1)
