Structural complexomics of eukaryotic gene expression
The Berger group studies eukaryotic multiprotein assemblies in transcription regulation, develops technologies to produce them recombinantly and subjects them to high-resolution structural and functional analyses.
Figure: We develop and utilise advanced, automated technologies to produce eukaryotic multiprotein complexes for structural and functional analysis by a variety of methods including X-ray crystallography.
Previous and current research
Human gene transcription requires the controlled step-wise assembly of the pre-initiation complex (PIC), comprising a large ensemble of proteins and protein complexes including RNA Pol II and the general transcription factors. TFIID is the first general transcription factor to bind to gene promoters, and is a cornerstone of PIC assembly. Understanding of TFIID and its crucial role in transcription regulation is hampered by a lack of detailed knowledge of its molecular architecture, assembly in the cell, and interactions with chromatin and other factors. The paucity and heterogeneity of TFIID in endogenous cells impede its extraction from cells for high-resolution structural and functional studies. Our lab addressed this challenge by creating new technologies for recombinant production of TFIID and other complex protein machines. Notably, our MultiBac system – a modular, baculovirus-based technology specifically designed for eukaryotic multi-protein expression – is now used in many labs worldwide, in areas including structural biology, vaccine development and gene therapy vectors. Recently, we determined the architecture of the 700 kDa heterodecameric human TFIID core complex by hybrid methods, combining MultiBac-based production, cryo-EM, X-ray crystal structures, homology models, and proteomics data. It is thought to represent a central scaffold that nucleates holo-TFIID formation and provided first impressions on how the functional holo-TFIID complex is assembled in the nucleus.
We collaborate with groups from academia and industry for technology development. We are striving to automate labour-intensive steps in the multiprotein complex structure determination process, and have harnessed homologous and site-specific recombination methods for assembling multigene expression plasmids. We have implemented a full robotics setup by developing ACEMBL, a proprietary automated suite for multigene recombineering on our TECAN EvoII platform. By using our technology, we produced numerous large multiprotein assemblies for structural studies, including multicomponent membrane protein complexes and the 1.6 MDa human TFIID holo-complex and expanded our multiprotein expression strategies to prokaryotic and mammalian hosts.
Future projects and goals
We continue to advance our expression technologies to entirely automate and standardise the process of production for eukaryotic gene regulatory multiprotein complexes including the entire human TFIID holocomplex, its various isoforms and other components of the preinitiation complex. In collaboration with the Schaffitzel Team and the Schultz Group (IGBMC Strasbourg), we subject the complex specimens produced to electron microscopic analyses. We use homogenous complexes thus identified for X-ray crystallography, aim to understand physiological function, and explore and challenge our findings by in vitro and in vivo biochemical analysis.
Using state-of-the-art mass spectrometric methods from systems biology, we are developing MultiTRAQ, a new technology addressing a further bottleneck in complex crystallography, namely the challenge of defining crystallisable core assemblies of multiprotein complexes in a reasonable time frame (a collaboration with ETH Zürich and Lund University). Another recent project line in our lab exploits synthetic biology techniques for genome engineering, with the aim of creating disruptive platforms for recombinant protein production, for both academic and industrial applications.