A research team of scientists from the EMBL Grenoble and the IGBMC in Strasbourg have now, for the first time, succeeded in describing in molecular detail the architecture of the central scaffold of human TFIID, made up of 10 proteins. This complex was previously identified in cell nuclei and is thought to represent the functional human TFIID core, onto which the remaining subunits assemble to give rise to the complete holo-TFIID complex. By applying innovative methods for recombinant protein production, developed and implemented at the EMBL, the scientists coaxed insect cell cultures which they infected with a custom designed baculovirus to produce their TFIID complex in the quality and quantity required for detailed studies. “Some time ago, we had developed our MultiBac system which uses a custom engineered recombinant baculovirus to express protein complexes, and many researchers all around the world applied it highly successfully to produce complicated biological samples that they could not make before.” says Imre Berger, who lead the study at the EMBL Grenoble. “However, for our human TFIID complexes, even this successful method was just not good enough.” The scientists realized that the insect cell cultures they infected with their recombinant baculovirus were simply unable to produce the TAFs in a balanced fashion. Instead, some were produced in high amounts, others in very low amounts, and the complex did not come together properly. The solution to this bottleneck came from studying the strategy certain viruses such as Coronavirus use to make their proteins. When Coronavirus replicates, it produces very large polyproteins that are then cut apart by a highly specific protease into the individual enzymes and protein factors that Coronavirus needs. Applying this strategy to producing TAFs with their MultiBac system lead to highly abundant and properly assembled complexes. These complexes could be purified and analyzed by hybrid methods combining high resolution cryo-electron microscopy, homology models and crystal structures of small parts of the TAFs, that had been determined before. “The quality of the images was exceptional.” confirms Gabor Papai who analyzed the micrographs using a high-end microscope at the IGBMC in Strasbourg. “This allowed us to determine the location of the proteins in the complex with previously unattainable accuracy.” This ground-breaking analysis reveals the inner workings of human TFIID for the first time, in unprecedented detail. The structure shows that parts of the TAFs adopt very defined structures, whereas other parts appear to adopt intricate, extended geometries winding like worms through the complex, holding it together. “There were numerous theories, based on scant data, trying to rationalize how this essential complex is held together” explains Christoph Bieniossek, first author of the study. “Our analyses show that basically none of these theories were correct. The way how the TAFs assemble tuned out to be much more complex than previously assumed.” The authors further describe how this TFIID core complex, which they found to have two-fold symmetry, becomes asymmetric when it grows by accreting further TAFs, on its way to the complete holo-TFIID. Two subunits, separately imported into the nucleus, bind exactly at the two-fold axis present in TFIID core, leading to large structural rearrangements but only in one half of the complex. By this simple yet elegant mechanism, the two halves of the complex which were previously identical adopt now different shapes, and the resulting asymmetry is then propagated until functional holo-TFIID complex is formed. “This work is the result of many years of intense effort, and it was only possible in this fruitful and exciting collaboration with our colleagues at the IGBMC who are leaders in human transcription factor research.” concludes Imre Berger. “We know now in some detail how the core of TFIID looks like, and what happens when further TAFs are bound. We believe that we have opened the door to working out the architecture of the entire human TFIID complex in the near future, and likewise the other large multiprotein assemblies involved in gene regulation, and explain their roles in catalyzing biological function.” The study, published in Nature today, involved the research groups of Imre Berger and Christiane Schaffitzel at the EMBL Grenoble, and the research groups of Patrick Schultz and Laszlo Tora at the IGBMC in Strasbourg. The MultiBac technology platform used in this study is a cornerstone of the EC FP7 ComplexINC project.