Max Planck Institute for Multidisciplinary Sciences
Dynamics and Energetics of Ribosomal Translation
The ribosome is a large biomolecular complex composed of ribonucleic acids and proteins. Ribosomes translate the genetic code (mRNA) into proteins and are therefore essential in all kingdoms of life. The ribosomal machinery operates on time and length scales which span several orders of magnitude. The aim of this project is to understand functional mechanisms of the ribosome at an atomic level using molecular dynamics (MD) simulations.
The transfer RNAs (tRNAs) carry amino acids and deliver them to the ribosome. Specific tRNAs carry specific amino acids and, by base-pairing to the mRNA presented by the ribosome, they translate the information. Deep within the ribosome, the new amino acid is incorporated into the growing peptide which leaves the ribosome through an exit tunnel. We develop an MD-based protocol to study the structure and dynamics of the elongating peptide within the exit tunnel.
After formation of the peptide bond between the new amino acid and the peptide chain, the tRNAs move through the ribosome. This tRNA movement is accompanied by movements of the ribosome including large-scale rotation of the small ribosomal subunit relative to the large subunit (Fig. 1b). MD simulations started from intermediate states resolved by cryo-EM allowed us to estimate transition rates for these motions suggesting that the intersubunit rotation is remarkably rapid and that the tRNA motion itself is rate-limiting [Bock 2013] (Fig. 1c).
The dynamic contact network between the subunits observed in the simulations results in an almost constant intersubunit binding enthalpy despite the large-scale relative rotations [Bock 2015]. A relatively constant affinity between the subunits for different rotation angles is a prerequisite for rapid rotation. In addition to the central intersubunit contact sites, peripheral contacts were found to maintain strong steady interactions, despite a large relative shift, by changing contact partners in the course of rotation.
During elongation, tRNAs are delivered to the ribosome in form of a ternary complex (TC), consisting of the tRNA, an elongation factor (either EF-Tu or SelB), and GTP. High-resolution cryo-EM structures of intermediates of the tRNA delivery to the ribosome uncover large-scale conformational changes of the ribosome and the TC [Fischer 2016]. To complement the structural information with energetics and rapid dynamics, we performed MD simulations of the free TC in solution. The TC was found to rapidly interconvert between the different conformations allowing us to construct the free-energy landscape of the involved motions (Fig. 3). This free-energy landscape indicates that the intrinsic large-scale conformational changes of the tRNA and SelB during the delivery to the ribosome are not rate-limiting to the process [Fischer 2016].
In some cases, the growing peptide chain in the exit tunnel leads to a stalling of the ribosome. In the case of the peptide ErmBL, the stalling in the presence of the antibiotic erythromycin is used as an erythromycin sensor which triggers expression of a resistance gene. Complementing structural information with MD simulations lead us to propose a stalling mechanism which involves a conformational change of the tRNA binding site resulting in a conformation unfavourable for the peptide bond formation [Arenz 2016].
Ribosome•EF-Tu complex stalled by antibiotic kirromycin
The atomistic model of the ribosome complex based on a cryo-EM structure [Fischer15] was used in simulations to investigate the mechanism of the antibiotics kirromycin [Warias2020] and the effect of cryo-EM cooling on structural ensembles [Bock2022]. Large and small ribosomal subunits are shown in transparent blue and orange. A-site, P-site and E-site tRNAs are shown in yellow, dark blue, and green. EF-Tu with bound antibiotic are colored in magenta and dark grey, respectively.
Kolar, M. H.; Nagy, G.; Kunkel, J.; Vaiana, S. M.; Bock, L. V.; Grubmüller, H.: Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel. Nucleic Acids Research 50 (4), pp. 2258 - 2269 (2022)
Beckert, B.; Leroy, E. C.; Sothiselvam, S.; Bock, L. V.; Svetlov, M. S.; Graf, M.; Arenz, S.; Abdelshahid, M.; Seip, B.; Grubmüller, H.et al.; Mankin, A. S.; Innis, C. A.; Vàzquez-Laslop, N.; Wilson, D. N.: Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics. Nature Communications 12, 4466 (2021)
Peng, B. Z.; Bock, L. V.; Belardinelli, R.; Peske, F.; Grubmüller, H.; Rodnina, M. V.: Active role of elongation factor G in maintaining the mRNA reading frame during translation. Science Advances 5 (12), eaax8030 (2019)
Fischer, N.; Neumann, P.; Bock, l. V.; Maracci, C.; Wang, Z.; Paleskava, A.; Konevega, A. L.; Schröder, G. F.; Grubmüller, H.; Ficner, R.et al.; Rodnina, M. V.; Stark, H.: The pathway to GTPase activation of elongation factor SelB on the ribosome. Nature 540 (7631), pp. 80 - 85 (2016)
Arenz, S.; Bock, L. V.; Graf, M.; Innis, C. A.; Beckmann, R.; Grubmüller, H.; Vaiana, A. C.; Wilson, D. N.: A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nature Communications 7, 12026 (2016)
Fischer, N.; Neumann, P.; Konevega, A. L.; Bock, L. V.; Ficner, R.; Rodnina, M. V.; Stark, H.: Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520 (7548), pp. 567 - 570 (2015)
Bock, L. V.; Blau, C.; Schröder, G. F.; Davydov, I. I.; Fischer, N.; Stark, H.; Rodnina, M. V.; Vaiana, A. C.; Grubmüller, H.: Energy barriers and driving forces in tRNA translocation through the ribosome. Nature Structural and Molecular Biology 20 (12), pp. 1390 - 1396 (2013)