Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.

We used single-particle electron microscopy to characterize the structure and subunit organization of the Mediator Head module that controls Mediator-RNA polymerase II (RNAPII) and Mediatorpromoter interactions. The Head module adopts several conformations differing in the position of a movable jaw formed by the Med18-Med20 subcomplex. We also characterized, by structural, biochemical and genetic means, the interactions of the Head module with TATA-binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7. TBP binds near the Med18-Med20 attachment point and stabilizes an open conformation of the Head module. Rpb4 and Rpb7 bind between the Head jaws, establishing contacts essential for yeast-cell viability. These results, and consideration of the structure of the Mediator-RNAPII holoenzyme, shed light on the stabilization of the pre-initiation complex by Mediator and suggest how Mediator might influence initiation by modulating polymerase conformation and interaction with promoter DNA.Transcriptional regulation is focused on the initiation process, which entails recruitment of RNAPII and the general transcription factors to a promoter. Both basal and activated transcription are critically dependent on the Mediator complex [1][2][3][4][5] , which conveys regulatory signals to RNAPII. Consistent with its essential role, the Mediator complex is conserved in sequence and structure throughout the eukaryotes [6][7][8] . Unfortunately, despite the paramount importance of Mediator, the mechanism of action of the complex remains unclear, highlighting the significance of investigating its structure, subunit organization and conformational variability.Biochemical and structural analyses have shown that Mediator has a modular organization. Biochemically defined subunit modules appear to correspond to structural modules identified by structural studies. Recent cryo-electron microscopy (EM) analysis of Mediator ( AUTHOR CONTRIBUTIONST.I., F.C. and Y.T. expressed, purified and biochemically characterized recombinant Head module and Head module subcomplexes and provided recombinant Rpb4 and Rpb7 and TBP; Y.T. and T.I. designed and carried out Head-Rpb4-Rpb7 and Head-TBP binding assays; K.Y. and Y.T. designed and carried out assays to test genetic interaction of Mediator subunits with Rpb4; Y.T. carried out in vitro transcription assays; G.C. carried out all EM data collection and analysis; G.C., Y.T. and F.J.A. discussed and interpreted all results; F.J.A. supervised EM structural analysis and wrote the manuscript in collaboration with G.C. and Y.T. COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Of all Mediator modules, the Head is perhaps the most critical, as evidenced by cessation of mRNA synthesis at nearly all promoters in vivo when Head module function is compromised in a Med17 temperature-sensitive Saccharomyces cerevisiae mutant strain [9][10][11] . Consistent with this obse...

SUMMARY Mediator, the multi-subunit complex that plays an essential role in the regulation of transcription initiation in all eukaryotes, was isolated using an affinity purification protocol that yields pure material suitable for structural analysis. Conformational sorting of yeast Mediator single particle images characterized the inherent flexibility of the complex and made possible calculation of a cryo-EM reconstruction. Comparison of free and RNA polymerase II (RNAPII)-associated yeast Mediator reconstructions demonstrates that intrinsic flexibility allows structural modules to reorganize and establish a complex network of contacts with RNAPII. We demonstrate that, despite very low sequence homology, the structures of human and yeast Mediators are surprisingly similar and the structural rearrangement that enables interaction of yeast Mediator with RNAPII parallels the structural rearrangement triggered by interaction of human Mediator with a nuclear receptor. This suggests that the topology and structural dynamics of Mediator constitute important elements of a conserved regulation mechanism.

Transportin 1 (Trn1) is a transport receptor that transports substrates from the cytoplasm to the nucleus through nuclear pore complexes by recognizing nuclear localization signals (NLSs). Here we describe four crystal structures of human Trn1 in a substrate-free form as well as in the complex with three NLSs (hnRNP D, JKTBP, and TAP, respectively). Our data have revealed that (1) Trn1 has two sites for binding NLSs, one with high affinity (site A) and one with low affinity (site B), and NLS interaction at site B controls overall binding affinity for Trn1; (2) Trn1 recognizes the NLSs at site A followed by conformational change at site B to interact with the NLSs; and (3) a long flexible loop, characteristic of Trn1, interacts with site B, thereby displacing transport substrate in the nucleus. These studies provide deep understanding of substrate recognition and dissociation by Trn1 in import pathways.

The five human RecQ helicases participate in multiple processes required to maintain genome integrity. Of these, the disease-linked RecQ4 is the least studied because it poses many technical challenges. We previously demonstrated that the yeast Hrq1 helicase displays similar functions to RecQ4 in vivo, and here, we report the biochemical and structural characterization of these enzymes. In vitro, Hrq1 and RecQ4 are DNA-stimulated ATPases and robust helicases. Further, these activities were sensitive to DNA sequence and structure, with the helicases preferentially unwinding D-loops. Consistent with their roles at telomeres, telomeric repeat sequence DNA also stimulated binding and unwinding by these enzymes. Finally, electron microscopy revealed that Hrq1 and RecQ4 share similar structural features. These results solidify Hrq1 as a true RecQ4 homolog and position it as the premier model to determine how RecQ4 mutations lead to genomic instability and disease.