The lab of Yuichiro Takagi, PhD (Indianapolis) is to functionally and structurally decipher molecular machines of gene regulation by development of new technologies and theories, and is particularly interested in the Mediator complex, a crucial component of transcription regulation in all eukaryotes.
Biology of Complexity: Deciphering complex molecular machines of gene regulation
Transcription by RNA polymerase II (Pol II) is fundamental to almost all biological activities. Thus, elucidation of transcriptional regulatory mechanisms is essential to understand biology and human diseases. The Mediator complex, or “Mediator”, is critical for Pol II transcription regulation. Mediator is a multi-protein complex composed of 25 subunits – a large molecular machine. Mutations in several human Mediator subunits have been linked to neurological disorders and cancers, exemplifying the importance of proper Mediator function for human health. Understanding how Mediator regulates Pol II transcription has been a major topic in the Takagi laboratory.
As a multi-protein complex, Mediator has been inherently difficult to decipher due to its sheer size, complexity and intricate network of intra-and inter-protein interactions. It has become clear that elucidating Mediator function is equivalent to solving a fundamental problem of biological complexity. While single-subunit proteins are often delineated in terms of their structure-function by their individual domain function, proper concepts or methods have yet to be developed to describe how the respective traits accumulate and achieve the molecular functionality of the complex. Thus, from a big picture point of view, the way complex molecular machines are thought of requires a new paradigm and approach, leading researchers toward definitive rules to describe the biochemistry of multi-subunit macromolecular assemblies and functions.
Developing Advanced Protein Complex Engineering Technology
One of the big technical problems when dealing with protein complexes such as Mediator is that an endogenous complex is often low-abundant and heterogeneous, limiting the ability to study them. Therefore, generating Mediator and other multi-protein complexes recombinantly is essential for structural and functional studies (cryo-EM and X-ray crystallography). Previously, the successful generation by the Takagi Lab of the recombinant Mediator Head module (7 subunits, 223 kDa), an essential sub-complex of Mediator, led to the structure determination of the complex by X-ray crystallography (see Figure). Further, the lab developed a unique baculovirus expression vector system that allows for the expression of large and problematic proteins, which are often found in molecular machines, in insect cells. This technology (termed SEP system) was the breakthrough that enabled the generation of Mediator, as well as other important proteins and protein complexes including plant RNA polymerase IV, DNA helicases, and RNA editing complex to name a few.
Determining Structure of Mediator Complex 21 Subunits
Mediator plays a critical role in transcription. Despite its importance, and the recent significant progress in structural studies, a high-resolution structure determination of the entire Mediator has not yet been achieved. The complexity (21 subunits), size (over 1 MDa), low abundance, and heterogeneity of native Mediator pose formidable challenges for structural studies. Development of the SEP system enables us to generate a functional a 21-subunit. Structural studies using cryo-EM are now underway. The pursuit of the structure determination of the entire Mediator by X-ray crystallography is a goal of the lab.
As important as it is for structure determination of Mediator (or other complexes), deciphering molecular mechanism of these complexes in vivo will be the ultimate goal. Multi-protein complexes are most likely multi-functional. Elucidation of each unique function in vivo by a traditional approach (e.g. mutagenesis) is not feasible due to the intricacy of complexes. To overcome this fundamental problem, the aim of the lab is to propose the development of a general method, enabling the lab to screen small compounds, which selectively inhibit or activate function(s) of the protein complexes. A chemical probe approach will help to dissect each unique function, thereby understanding overall functions of the complex. The work of the lab aims to screen for small compounds by leveraging the ability to generate high-value proteins and protein complexes (e.g. drug targets) in large quantities. Some of the chemical probes will most likely become lead compounds prompting a possible drug discovery.