The overall objective of this research project is the elucidation of the process of translation (i.e., translation from genetic information into protein) in eukaryotes, including mammals. Although X-ray structures exist for a number of eukaryotic ribosomes, the structural basis of this process has been mainly investigated by cryo-electron microscopy (cryo-EM). However, many short-lived states, with a life time of less than a second, cannot be imaged by standard cryo-EM (characterized by pipetting/blotting of sample), leaving large gaps in our understanding of these basic processes of life and in the knowledge base important for molecular medicine. In the approach of time-resolved cryo-EM adopted in this lab, reactions are started in a microfluidic chip (silicon- or plastic-based) by mixing two components, letting them react for a defined time (10 to 1000 ms) determined by flow rate and length of reaction channel, and the reaction product is sprayed on the EM grid immediately before the latter is plunged into the cryogen (liquid ethane, on the temperature of liquid nitrogen). In the current renewal period of this grant, ending on March 31, 2019, this technique has been greatly improved, and applied to three processes important in bacterial translation: initiation, termination, and ribosome recycling. All three applications have been successful, resulting in the capture of a short-lived state at close-to-atomic resolution and leading to papers either published or under consideration. For the current renewal, exploration of short-lived states in eukaryotic translation will have even greater relevance for human health. In collaborations with leading experts in eukaryotic translation, the Frank Lab team will apply time-resolved cryo-EM to the elucidation of processes during eukaryotic translation initiation, termination, and recycling.
The aim to determine atomic structures for short-lived states that are impossible to capture by standard cryo-EM. Using these structures along with those obtained from conventional cryo-EM and X-ray crystallography, the time courses and pathways taken in the respective processes can be modeled for the first time, based on solid experiments. This new knowledge will advance strategies for combatting many diseases that implicate dysfunctions of eukaryotic translation.
There is growing awareness not only that translation (of the genetic message into proteins) is central to processes that determine human health and development, and that defects in translation or its regulation underlie many human diseases, but also that steps in translation constitute targetable vulnerabilities. Inhibitors of the elongation have been studied in detail, and it is therefore important that molecular mechanisms of less well- characterized stages of the translation process are elucidated, as well. This project aims to focus on short-lived states during translation initiation, termination, and recycling in eukaryotes, states that are impossible to visualize with X-ray crystallography or standard cryo-EM.
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