Phone: (414) 955-5849
Fax: (414) 955-6510
Structure of the 12-subunit RNA polymerase II refined with the aid of anomalous diffraction data.
This figure shows the most complete structure of RNA Polymerase II (Pol II), the enzyme that reads genetic sequences from chromosomes in the nucleus of a cell. The Pol II model is rendered in the form of ribbons (most of it in blue) and laid over a background image of chromosomal DNA. The structural work was performed in the Fu lab by utilizing the power of anomalous scattering from 8 Zn2+ ions bound intrinsically in the polymerase. This latest model reveals the previously undetermined loop (green) that is implicated in contacting the general transcription factor TFIIF, and defines conformation for the loop (orange) that crosslinks with TFIIE. Several functionally significant elements are highlighted as follows: yellow, Fork Loop-1; grey-brown, Fork Loop-2; pink, Rudder and red (sphere), the catalytic Mg2+ site. The chromosomal DNA is depicted for its unraveling at the different levels, from the sister chromatids to 30-nm chromatin fiber and 10-nm nucleosomal array.
This work is described in: Meyer PA, Ye P, Suh MH, Zhang M, Fu J., J Biol Chem. 2009 Mar 16. [Epub ahead of print]
Current research in the laboratory is focused on the structural mechanisms of gene transcription and regulation in eukaryotic cells. Transcription is the first step in the expression of genetic information from a cell's chromosomal DNA. In eukaryotic cells, three RNA polymerases, named Pol I, II and III, carry out transcription. Among them, Pol II is responsible for synthesizing protein-coding RNAs, the messenger RNAs (or mRNAs) that direct the synthesis of cellular proteins. Transcription by Pol II is one of the fundamental processes that underlie development, oncogenesis and viral pathogenesis (e.g. HIV-AIDS).
The Pol II transcription system forms a multi-protein machine with the RNA polymerase at its heart and other accessory protein factors around it. Many components of this gigantic apparatus have been discovered over the past 40 years, but understanding is still missing as to how this machine controls its initiation frequency and its RNA synthesis rate, in response to external signals. To gain insights into its mechanism, detailed knowledge of the 3-dimensional (3-D) workings of the machinery is required. Owing to the breakthrough in determining the structure of the free Pol II (Fu et al., Cell, 98: 799-810, 1999; Cramer et al., Sci., 288: 640-649, 2000), it is now feasible to determine the 3-D structure of the apparatus in more extensive fashions. Since Pol II by itself is dormant, we are taking steps to work out Pol II-factor(s) complex structures that correspond to its active functions in vivo.
We are particularly interested in post-initiation steps in the transcription cycle (Figure). Intermediate Pol II-factor complexes involved in this stage of transcription have been shown to be targets of viral activators, restrictor of estrogen alpha-dependent growth of breast epithelial cells, and regulators of gene activities in response to environmental cues. We are working on cocrystal structures of complexes formed between Pol II and factors known to be involved in this process.
Figure: A simplistic model for Pol II initiation, early elongation and reinitiation. Pol II and TBP-TFIIB-TATA promoter complex are each rendered as solvent accessible surfaces. PIC stands for the pre-initiation complex. Cet1/Ceg1 represents the pre-mRNA capping enzyme. DSIF is the negative elongation factor and, pTEFb, the positive elongation factor. Protein-protein interactions within the early elongation complex (EEC) are indicated by two-way arrows. The CTD domain of the largest subunit of Pol II is represented by the zigzag line. Fcp1 is a CTD phosphatase, plays a key role in generating initiation-competent Pol II for reinitiation.
We take a multidisciplinary approach to analyze 3-D structures of large complexes. We use Protein Biochemistry and Molecular Cloning to obtain pure materials and assessing their activities. We then apply X-ray Crystallography to determine their 3-D structures. X-ray crystallography (figures below) is the primary method for determining atomic structures of biological macro-molecules. Computational techniques are being continually improved in the lab to cope with the complexity of large molecular systems such as complexes of RNA polymerase II. Recent developments in X-ray detector technology and synchrotron instrumentation have greatly facilitated our structural work.
The department houses state-of-the-art instrumentation dedicated to Structural Biology research. The facility includes chromatographic systems for protein purification, an in-house X-ray diffraction core and an automated crystallization machine for high-throughput screening and optimization. High-end computer workstations have been set up for 3-D graphic visualization and fast-speed crystallographic analysis.
Graduate students and other research members of the lab receive systematic training in Structural Biology in general, and X-ray Crystallography and Protein Biochemistry in particular. This lab believes in logic, rigor and the validation of scientific results. Training in this lab usually entails instruction, discussion and exploration: from conceiving ideas, designing approaches for testing hypotheses, analyzing complex observations and experimental data, integrating information, to gaining scientific insights.