To study the dynamic super-assemblies of multiple ribosomes, mRNA strands, chaperones, and nascent chains, we have proposed a program that gradually increases in complexity. It starts with the elementary interactions between participating components; the ribosomes (Aim 1) and the nascent chains (Aim 2), and moves progressively towards the most elaborate systems (Aim 3).
In an effort to efficiently produce stalled nascent chain complexes for both soluble and membrane proteins described under aims 1, 2 and 3, the ETH team optimised human in vitro translation system for efficiency and ease of sample preparation. The paper describing our findings was recently published in Cell Reports Methods (Bothe and Ban, 2024). This system has now been shared across all groups and used in collaborative experiments.
Aim1: During the first year, the Heidelberg team has elucidated the genome-wide pattern of co-co homodimer assembly (involving interaction of two nascent chains) by generating deeply sequenced disome profiles in bacteria. These data sets we have used to determine the prevalence for cytosolic and membrane protein complexes and for comparing assembly onsets with emergence of dimerization domains (Aim1). These data furthermore yield representative examples of the classes, which we will study at the molecular level using single-molecule and cryo-EM methods (see next tasks). Furthermore, we focus on establishing our new genome-wide disome profiling method DiSPRITE to explore if co-co assembly occurs in cis, if ribosomes are adjacent at assembly onset and how their distance evolves during translation.
The ETH team is closely collaborating with the Heidelberg group to investigate co-translational assembly of the Escherichia Coli chaperonin GroEL. The fully assembled chaperonin forms two stacks of heptameric homo-oligomers, resulting in a large tetradecameric complex. To complement the biochemical insights obtained in the Bukau group we are using cryo-electron tomography to investigate the organisation of ribosomes in the context of polysomes that are in the process of synthesizing GroEL. To be able to visualise the organisation of ribosomes in the bacterial cell using tomography we first need to prepare an extremely thin lamella of the cell using a focused ion beam microscope (FIB), which allows us to very precisely remove material by focusing a beam of plasma or ions on selected regions of the sample. These procedures are technically difficult and we are currently establishing methods to prepare such lamellae in a reproducible and high-throughput manner.
Aim 2: The Heidelberg group has determined binding profiles of co-translationally acting chaperones in bacteria and human cells and has developed a new proteome-wide approach to explore the prevalence of co-post assembly (involving interaction of a nascent chain and a fully synthesized partner protein). The method was established in bacteria and will be adapted for use in human cells. To reveal new co-factors and coordination mechanisms of the assembly process, genome-wide screens are being performed in human cells to identify novel regulatory factors that guide the assembly process. For mechanistic analysis of factors, in vitro ribosome profiling (DiSP, SeRP) is currently established.
Aim 3: To dissect co-translational assembly in systems of increasing complexity (membrane complexes, alternative compartments, higher order complexes), the Heidelberg team has focussed on establishing multiple new methods, including an alternative method to DiSP that can be used to study membrane complex assembly, the DiSPRITE and another novel multi-ribosome tagging system.
The Ban laboratory is currently studying how co-translational interactions between partially synthesised nascent chains facilitate membrane protein complex formation at the endoplasmic reticulum (ER) membrane. Our aim is to determine the composition and higher order organization of ribosome-nascent chain complexes (RNCs) involved in co-translational assembly at the ER membrane. To this account we prepared ER vesicles from human cells overexpressing several model nascent chains, and imaged them after purification by single particle cry-electron microscopy or by cryo-electron tomography. We are also optimizing the pipeline for subtomogram averaging of ER-bound ribosomes. This has led to reconstructions of ER-bound ribosomes in the context of an ER-bound polysome with additional ER luminal features that can be assigned to known Sec61 translocon-binding factors. We are also developing computational analysis methods with an aim to determine the relative position and orientation of ribosomes in order to detect patterns in their cellular organization.
