Visual Translatomics – Deep ribosomal insights by cryo-electron microscopy of actively translating ribosomes | DeepRibosome | Projekt | Informationsblatt | HORIZON | CORDIS

Projektbeschreibung

Enthüllung des Ribosoms in Bewegung

Ribosomen sind die molekularen Maschinen des Lebens. Sie sind wie winzige Fabriken, die den genetischen Code in Proteine übersetzen. Doch trotz jahrzehntelanger Studien sind ihre schnellen und dynamischen Bewegungen nach wie vor schwer zu erfassen. In diesem Zusammenhang zielt das vom Europäischen Forschungsrat finanzierte DeepRibosome-Projekt darauf ab, eine neue Methode zu entwickeln, um Ribosomen bei ihrer Arbeit in Echtzeit zu beobachten. Konkret wird die Kryo-Elektronenmikroskopie an aktiven, aus Zellen stammenden Ribosomen eingesetzt, die in einem kontrollierten System rekonstituiert wurden. Das Projekt wird flüchtige strukturelle Zustände abbilden, die in wenigen Millisekunden auftreten. In Verbindung mit funktionellen Assays wie Ribo-seq wird dieser Ansatz Struktur und Funktion mit höchster Präzision verbinden. DeepRibosome verspricht Aufschluss darüber zu geben, wie Ribosomen sich anpassen, die Translation regulieren und auf ihre Umgebung reagieren, selbst unter Antibiotikastress.

Ziel

Structure determination of dynamic macromolecular molecular machines and elucidating their conformational landscapes are fundamental tasks of structural biology. The ribosome as a paradigmatic macromolecular machine interacts with numerous ligands and factors throughout its functional cycles, which in turn are driven and regulated by various conformational modes. Project DeepRibosome proposes a new way to perform structural studies. It is based on our recent finding that E. coli ex-vivo derived polysomes carrying the translatome can be efficiently reactivated in the PURE in vitro translation system. Cryo-EM of actively translating ribosomes results in comprehensive structural landscapes. During steady state, all intermediates are expected to be present, including the short-lived ones. Supported by preliminary data, we estimate that applying multi-particle sorting of images we can resolve states with a lifetime in the one-digit millisecond range. This allows us to study under optimal conditions fast steps such as the GTPase factor-dependent reactions or peptidyl-transfer/accommodation without artificially stalling the reaction by antibiotics or non-cleavable GTP analogues. The structural studies will be accompanied by functional assays including Ribo-seq to monitor translation of the cell-derived translatome in a cell-free system. While, we will also conduct in situ structural analysis for comparison, the strength of our new approach is that we can track translation from an integrated structural and functional perspective, preserving/reconstituting many native features but under defined and controllable conditions. This will not only provide insights into the molecular mechanism of translation at the next level of understanding but also allow us to answer long-standing questions e.g. how exactly the physicochemical environment (temperature, ions) or auxiliary factors impact translation. It will also provide new means to study the inhibition mechanisms of antibiotics.