Structure and Mechanism of the human Transcription Machinery
The Taatjes lab investigates the molecular mechanisms by which the human transcription machinery functions and is regulated. Proper regulation of gene expression is fundamental to every major physiological process, and changes in gene expression patterns are hallmarks of human development and disease. Consequently, the questions that we address in the Taatjes lab are fundamental and of broad significance. At the moment, our research has direct implications for cardiac and neuronal development, inflammation, cancer, and aging.
The macromolecular assembly required to initiate gene expression consists of 8 protein complexes and is approximately 4.0 MDa in size. The human Mediator complex (26 subunits, 1.2 MDa) is a major sub-assembly within the transcription apparatus and represents the main focus of the lab. Although the mechanisms by which Mediator functions to control gene expression are poorly-defined, it is clear that Mediator is essential for expression of most protein-coding and non-coding RNA genes and regulates gene expression in myriad ways, including transcription initiation, elongation, RNA processing, and chromatin architecture. Mediator is generally targeted by DNA-binding transcription factors that control gene expression programs in response to developmental and environmental cues. Furthermore, the Mediator complex interacts directly with the RNA polymerase II (pol II) enzyme; thus, Mediator functions generally by communicating regulatory signals from DNA-binding transcription factors to the pol II enzyme. Mediator remains poorly understood in part because Mediator structure and even its composition can change, depending upon the context. For example, DNA-binding transcription factors alter Mediator structure upon binding the complex, and a four-subunit, 600 kDa “CDK8 module” can reversibly associate with Mediator and change its biological function.
In the Taatjes lab, we utilize an array of diverse experimental approaches and have the privilege of working with a talented group of collaborators. Techniques employed range from basic biochemistry (protein expression and purification, enzymatic assays, in vitro transcription), mass spectrometry (LC-MS/MS, linear ion-trap MS, SILAC, CXMS), structural biology (cryo-electron microscopy, X-ray crystallography and/or NMR), metabolomics, cellular assays, and genomics (ChIP, RT-qPCR, RNA-Seq, microarray, ChIP-Seq). We have biochemically purified each of the factors that, together with Mediator, comprise the fully functional, 4.0 MDa human transcription apparatus (which includes RNA polymerase II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH). This enables us to address specific mechanistic questions regarding Mediator’s role as a regulator of pol II transcription.
