Time-resolved cryo-EM studies of translation initiation – TREMTI

In all living cells, translation initiation allows accurate selection of the start codon on a messenger RNA, establishing the reading frame of the protein to be synthesized. This essential step universally involves a macromolecular initiation complex (IC) containing the mRNA, the small ribosomal subunit, a methionyl-initiator tRNA, and translation initiation factors (IFs). Four of the IFs play essential roles in both eukaryotes and archaea. While developments in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided a wealth of new structural information, they have also raised many new questions regarding the dynamics of the initiation process. We propose to elucidate several key events and intermediates and the nature of the dynamical transitions between them. We will use a combination of structural, biochemical and computational tools, applied to archaeal systems. We will exploit recent advances in cryo-EM and achieve further methodological advances that will be of general interest.
We will solve several structures of initiation complexes or sub-complexes describing snapshots of the whole process. Importantly, to better describe the system dynamics, the ICs will be studied by cryo-EM not only at equilibrium, but at the pre-steady state, frozen at very short times after mixing. The idea that time-resolved cryo-EM has the potential to depict short-lived states of molecular complexes has emerged only recently. We will help lift several technical barriers by designing microfluidic devices for time-resolved cryo-EM and developing cutting-edge methods for fitting/refining 3D models into cryo-EM maps. These tools will be made available to the whole structural biology community. Experimentally-determined structures of both stable and short-lived intermediates will be supplemented by realistic molecular dynamics simulations that explore the complexes and transitions between some of them. Along with biochemical experiments, the whole approach will allow us to derive valuable new information on the role of dynamics in driving translation initiation.
The project will have a broad impact, since regulation of translation initiation plays crucial roles in processes as diverse and important as cellular proliferation, apoptosis, antiviral and antitumoral responses, sensing of amino acid deficiency in the brain and long-term potentialization of synapses. Archaeal initiation is of interest per se but also for its potential to provide new understanding of eukaryotes. Thus, recent studies of archaeal aIF2 revealed the structural basis of an important human genetic disease. More generally, our project will help characterize important potential drug targets, and contribute to our understanding of a fundamental process of life.