Protein Cell 2013, 4(12): 911–920 DOI 10.1007/s13238-013-3069-y Protein & Cell RESEARCH ARTICLE Structural fl exibility and functional interaction of Mediator Cdk8 module Xuejuan Wang1*, Jianye Wang1*, Zhenrui Ding1, Jinhua Ji1, Qianqian Sun1, Gang Cai1,2 1 School of Life Sciences, University of Science and Technology of China, Hefei 230026, China 2 Hefei National Laboratory for Physical Sciences at the Microscale, Center for Integrative Imaging, Hefei 230026, China Correspondence: [email protected] Received August 2, 2013 Accepted August 26, 2013 Cell ABSTRACT effi ciency by interacting with regulatory DNA. Mediator, a large & protein complex (>1000 kDa) comprising 25 different proteins, Mediator is a highly conserved large protein complex (25 is preeminently responsible for PIC assembly and conveying proteins, >1000 kD a) and preeminently responsible for eu- regulatory signals from DNA-binding transcription factors to karyotic transcription, which contains a dissociable ‘Cdk8 the PIC (Malik and Roeder, 2000; Naar et al., 2001; Kornberg, module’. Although increasing evidence demonstrates that 2005). Mediator is organized structurally and functionally in Protein Cdk8 module plays both positive and negative roles in three core modules (Head, Middle and Tail module) and a dis- transcription regulation, the detailed structure, and subu- sociable ‘Cdk8 module’ consisting of the cyclin-dependent ki- nit organization, molecular mechanism how it regulates nase Cdk8, its partner CycC, Med12, and Med13 (Borggrefe et transcription remain elusive. Here we used single-particle al., 2002; Chadick and Asturias, 2005). Core Mediator contains electron microscopy to characterize the structure and three stable modules: Head, Middle, and Tail, while the disso- subunit organization of the Cdk8 module and illuminated ciable Cdk8 module dynamically interacts with core Mediator. the substantial mobility of the Med13 subunit results in Understanding both the structure and regulation of the Me- the structural fl exibility. The Cdk8 module interaction with diator complex is essential to elucidate the mechanism of gene core Mediator is concurrent with active transcription in regulation (Chadick and Asturias, 2005; Lariviere et al., 2012). vivo. An interaction with the Cdk8 module induces core Our understanding of Mediator assembly and its role in regu- Mediator into very extended conformation in vitro, which lating transcription has been impeded so far by the large size, is presumed to be an active functional state of Mediator. low abundance, dynamic composition, and intrinsic fl exibility of Taken together, our results illuminated the detailed archi- Mediator, which has been a formidable challenge for structural tecture of Cdk8 module, and suggested the Cdk8 module analysis. Single particle electron microscopy (EM) is uniquely could positively regulate transcription by modulating Me- suited for structural analysis of the large multi-protein com- diator conformation. plexes and previous EM studies have contributed signifi cantly to understanding functional architecture of Mediator (Asturias KEYWORDS Mediator, Cdk8 kinase module, transcription et al., 1999; Dotson et al., 2000; Taatjes et al., 2002; Cai et al., regulation 2009). The most detailed cryo-EM structure of yeast core Me- diator highlights the remarkable conformational fl exibility and reveals its modularity features (Head, Middle, Tail, and Arm) INTRODUCTION (Cai et al., 2009). Not only acquiring the structural information, Regulation of eukaryotic transcription is largely focused on single particle EM is ideally suited to characterize the confor- the transcription initiation process, which involves assembly mation of Mediator under different conditions and illuminate of the large pre-initiation complex (PIC). Typically the PIC is how it is affected by interaction with various factors. Under- composed of RNA polymerase II (Pol II) and six general tran- standing the interaction of Mediator with the general RNAPII scription factors (GTFs) including IIA, IIB, IID, IIE, IIF, and IIH machinery has derived from EM structures of the Mediator- (Lee and Young, 2000). Transcription factors (including acti- RNAPII complex (Davis et al., 2002; Bernecky et al., 2011), vators and repressors) control the transcription initiation and Mediator-TFIIH complex (Lee et al., 2010), Mediator Head- *These authors contributed equally to the work. © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 December 2013 | Volume 4 | Issue 12 | 911 RESEARCH ARTICLE Xuejuan Wang et al. A B C Yeast whole cell extraction MW (kDa) 200 Figure 1. Preparation and Med12/SRB8 150 EM characterization of the Ammonium sulfate cut Med13/SRB8 120 Mediator Cdk8 module. (30%–55%) 100 85 (A) Scheme for affi nity pu- rification of Mediator Cdk8 70 Cdk8/SRB10 module from the whole cell IgG sepharose 60 extraction of tagged S. cer- TEV protease 50 evisiae cells. (B) Ruby-stain digestion SDS-PAGE analysis of pu- Mono Q 40 rified yeast Cdk8 module. (C) A micrograph showing CycC/SRB11 0.5 mol/L AS single Cdk8 module par- 30 ticles preserved in uranyl 0.1 mol/L formate. Scale bar, 100 nm. 20 Cdk8 module 10 Cell & TBP (Cai et al., 2010), and Mediator Head-RNAPII (Cai et al., appears to harbor important functional implications. Moreover, 2010, 2012). we characterized, by structural and biochemical means, the The cyclin-dependent kinase Cdk8, its partner CycC, interactions of Mediator with the Cdk8 module and RNAPII. Med12, and Med13 form a dissociable ‘Cdk8 module’, which Surprisingly, Mediator harbors strong interaction with RNAPII is critical for transcriptional regulation. Notably, Cdk8, a com- during transcription inactive stationary phase and only minor Protein ponent of Mediator, is an important oncogene (Firestein et al., binding with RNAPII in transcription highly active exponential 2008; Adler et al., 2012). Two previous publications on EM phase. In contrast, Cdk8 module prefers to strongly interact structural studies on Cdk8 module from fi ssion yeast and hu- with Mediator during exponential phase, which is concurrent man have reported it interact differently with Mediator in the with active transcription, thus suggesting Cdk8 module is in- two species and could repress transcription through either oc- volved in transcription activation. In vitro, both purifi ed RNAPII cupying the RNAPII binding sites or locking the conformation and Cdk8 module could dynamically bind to the Mediator in- of Mediator to prevent RNAPII binding, respectively (Elmlund dependently of other factors. The substantial conformational et al., 2006; Knuesel et al., 2009a). However, the detailed changes of Mediator upon interaction with RNAPII and Cdk8 structure and subunit organization of Cdk8 module, the precise module is comparable and Mediator was induced to adopt ex- mode of interaction between Cdk8 module and Mediator, and tended conformation where Middle and Tail module move far the mechanism of regulation by Cdk8 module remain unclear. apart. These results shed light on the regulation of transcription Moreover, instead of the established repressive role in tran- by Cdk8 module and suggest how Cdk8 could infl uence tran- scription (Holstege et al., 1998; Myers et al., 1998; Taatjes et scription by modulating Mediator conformation. al., 2002), the Cdk8 module has been increasingly substanti- ated that could also contribute to transcription activation (Be- RESULTS AND DISCUSSION lakavadi and Fondell, 2010; Donner et al., 2010; Conaway and Purifi cation of the Mediator Cdk8 module from Conaway, 2011). Therefore, we are interested to study struc- S. cerevisiae ture, subunit organization, and functions of Cdk8 module in more detail to achieve a comprehensive understanding of the Mediator is a low abundance, fragile, and large protein com- Cdk8 module’s function in transcription regulation. plex (>1000 kDa) (In S. cerevisiae, comprising 25 different We used single-particle electron microscopy, along with bio- proteins), which posed a serious impediment for biochemical chemical assays to characterize the structure and subunit or- preparation. We have established a novel affi nity purifi cation ganization of the Cdk8 module from Saccharomyces cerevisi- method (Fig. 1A) that readily and reproducibly yields highly ho- ae (S. cerevisiae). When we are preparing the manuscript, the mogeneous and functional yeast core Mediator complex (dis- structure of the yeast Cdk8 module has been published online sociable Cdk8 module is absent), which has resulted in the fi rst (Tsai et al., 2013) and some of the data of the subunit organi- cryo-EM reconstruction of Mediator (Cai et al., 2009). Moreo- zation of Cdk8 module presented in this paper are consistent ver, we also successfully applied the method to purify the Cdk8 with our fi ndings. However, our EM structure, for the fi rst time, module (Fig. 1B). To further remove some minor contamina- revealed the Cdk8 module adopts several different conforma- tions, an ion exchange Mono Q column is employed to polish tions corresponding to the mobility of the Med13 subunit, which the purifi cation. The improved procedures yielded the complete 912 | December 2013 | Volume 4 | Issue 12 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 Mediator Cdk8 module RESEARCH ARTICLE A B Figure 2. Structural flexibility of Cdk8 module. (A) Four different conformations of the Cdk8 module were identifi ed through reference-free alignment and classifi cation of EM im- I II ages of particles preserved in stain. I II 1094 797 Front The number