Project Details
Projekt
Translational GTPases and the energy landscape of the 70S ribosome
Applicant
Professor Dr. Christian M. T. Spahn
Subject Area
Structural Biology
Term
from 2012 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 207100805
Translational GTPases (trGTPases) constitute an important group of external translation factors. They control and steer the ribosome during all four phases of protein synthesis. trGTPases usually bind to a specific state of the ribosome in their GTP conformation and dissociate in their GDP conformation. Consequently, there are complex dynamic molecular interactions and interdependences during the functional cycle within the various intermediates. GTP hydrolysis and phosphate release may be critical steps in defining the molecular interplay with the ribosome. In the metastable energy landscape view the ribosome can be regarded as a Brownian machine capable of sampling several conformational states at ambient temperature.trGTPases can tune the energy landscape resulting in apparent large-scale conformational changes in the ribosome or the factor by a conformational capture mechanism. Here we want to structurally analyze the complex interplay between canonical translational GTPase factors with the bacterial 70S ribosome using multiparticle cryo-EM. In the second funding period the work on tRNA selection and translocation and the respective trGTPase elongation factors EF-Tu and EF-G shall be continued. Furthermore, we will include also the translational GTPases IF2 and RF3 involved in translation initiation and termination, respectively to facilitate a comprehensive overview of all four universally conserved trGTPases. We will capitalize on the advent of direct electron detection cameras for cryo-EM that now makes it feasible to obtain cryo-EM maps of ribosomal complexes at near-atomic resolution. Hereby the continued technical collaboration among the project using cryo-EM (P2, P3, P4) will be of crucial importance. This will allow us to solve the structure and conformational modes of various complexes of the 70S ribosomes with a trGTPase stalled on the ribosome in a variety of experimental conditions, e.g. using antibiotics or non-hydrolysable GTP analogues. Experiments on initiation complexes containing IF2 and other initiation factors will be done in close collaboration with P6. Functional studies and further support from P5, P6 and P7 will be required for an interpretation of the structural work in a functional context. The comparison of results for canonical GTPase factors with studies on non-canonical GTPase factors (P2) will yield important insights into the evolutionary conserved and divergent features of translational GTPase factors. Furthermore, the analysis on translationally paused ribosome-bound nascent chains by P7 will be supported by structural studies.
DFG Programme
Research Units
