The protein of interest is an essential component of the cleavage and polyadenylation factor IA (CF IA) and is involved in pre-mRNA 3’ end processing and in transcription termination. This protein is a multi-domain protein that interacts with a C-terminal domain (CTD) of RNA polymerase II (RNAPII) via one of its domain, namely CTD-interacting domain (CID). The goals of the thesis are:

1. To identify all domains of the protein responsible for RNAPII binding and forming biologically relevant structures
2. To optimize and to express the constructs from 1)
3. To express and to purify 15N and/or 13C isotopically enriched samples in amounts and quality need for structural studies using nuclear magnetic resonance (NMR) spectroscopy
4. To perform NMR titration with the aim to localize interacting surface and reconstitute stoichiometric complex
5. To acquire, process and analyse NMR datasets for assignment of resonance frequencies of backbone and side-chain resonances
6. To determine 3D structure of all the necessary components with the use of NMR, small angle X-ray scattering (SAXS), and/or X-ray crystallography
7. To perform fluorescence anisotropy (FA) assay that will confirm relevance of the determined structure
8. To refine the structure with AMBER software and validate the final structure using standard protein DataBank (PDB) protocols

Within this diploma thesis, the student will get acquainted with modern techniques used in structural biology and biophysical characterization of protein characterization and protein-protein interaction.
References:

• Xu, X., Pérébaskine, N., Minvielle-Sébastia, L., Fribourg, S., Mackereth, C.D.: Chemical shift assignments of a new folded domain from yeast Pcf11. Biomol NMR Assign. 2015 ;9(2):421-5
• Amrani, N., Minet, M., Wyers, F., Dufour, M.E., Aggerbeck, L.P., Lacroute, F.: PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I. Mol Cell Biol. 1997;17(3):1102-9
• Barillà, D., Lee, B.A., Proudfoot, N.J.: Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):445-50.
• Ni, Z., Xu, C., Guo, X., Hunter, G.O., Kuznetsova, O.V., Tempel, W., Marcon, E., Zhong, G., Guo, H., Kuo, W.H., Li, J., Young, P., Olsen, J.B., Wan, C., Loppnau, P., El Bakkouri, M., Senisterra, G.A., He, H., Huang, H., Sidhu, S.S., Emili, A., Murphy, S., Mosley, A.L., Arrowsmith, C.H., Min, J., Greenblatt, J.F.: RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation. Nat Struct Mol Biol. 2014 21(8):686-95.

The thesis will be written in English or Czech language