Figure 6: Nuclear TFIID assembly from preformed submodules.
(a) A complex consisting of TAF2, TAF8, TAF10 and Importin α1ΔIBB was formed from highly purified components. Importin α1ΔIBB was mixed in a twofold molar excess with purified TAF2–8–10 and the mixture purified by SEC. SDS–PAGE analysis of the peak fraction is shown. (b) Importin α1-TAF8 complex crystal structure. Magnified view of interacting residues of the major binding site of Importin α1 (grey) with residues of the NLS of TAF8 (blue). Importin α1 is shown in ribbon representation and the TAF8–NLS as a stick model. TAF8 residues R303 and R304 are not involved in contacting Importin α1 and are omitted for clarity. (c) Immunofluorescence microscopy of HeLa cells depleted of TAF8 (TAF8 siRNA) by RNAi or control cells (Control siRNA). Nuclei are visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). TAF2 is displayed in green and TAF8 in red. Arrows point to a non-transfected cell. Scale bar, 10 μm. (d) Cartoon model of cytoplasmic TAF2–8–10 complex and nuclear holo–TFIID assembly. The NLS of TAF8 is filled in black. The TAF2-interaction domain within TAF8 is highlighted by shading. TAF2, blue; TAF8, green; TAF10, orange. The TAF2–8–10 complex resides in the cytoplasm, whereas the physiological symmetric core–TFIID complex is found in the nucleus10,22. The cryo-electron microscopy density envelope of core–TFIID complex is shown (adapted from ref. 12). On binding of Importin α1 (grey) to the TAF8 NLS, TAF2–8–10 translocates into the nucleus through a nuclear pore (arrow). In the nucleus, Importin α1 is released and TAF2, 8 and 10 associate with core–TFIID, to form intermediates including the asymmetric 8TAF complex along the holo–TFIID assembly pathway.
