Mechanisms regulating speed and accuracy of translation

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Speed and accuracy of protein synthesis are crucial for the survival of cells and their adaptation to changing environmental conditions. During ongoing translation, periods of rapid synthesis are interrupted by pauses; the detailed nature and duration of those pauses remains poorly understood. In this project we propose to analyze translation pauses by a combination of time-resolved high-performance in-vitro translation assays and stochastic modeling. One special case is translation pausing when strings of proline residues are to be incorporated. This stalling is alleviated by elongation factor P (EF-P). We will study why stretches of prolines inhibit the peptidyl transferase activity of the ribosome and how EF-P rescues the ribosome from stalling by measuring the rates of synthesis of proline-rich sequences using a toolbox of proline analogs and comparing the chemical properties of the analogs with the efficiency of translation. Translation delays in specific regions of the mRNA may facilitate co-translational folding. We will follow the synthesis, co-translational movement of nascent peptides through the exit tunnel of the ribosome, and their folding by time-resolved FRET (Förster Resonance Energy Transfer) and PET (Photoinduced Electron Transfer) methods using co-translational insertion of non-canonical amino acid derivatives. The structure of the complexes during movement through the tunnel and folding will be solved by cryo-EM and modelled by molecular dynamic simulations.To understand the balance between the speed and accuracy of protein synthesis, we plan to systematically evaluate the error frequencies of protein synthesis in vivo in bacteria using mass spectrometry. We will determine which errors are prevalent, how cells respond to sub-lethal doses of error-inducing antibiotics and how the proteome responds to high error loads. Function and structure of two translational GTPases (SelB and IF2) will be studied. The project will show how the speed and accuracy of protein synthesis contribute to the quality of the cellular proteome.