Structural Study Of The Interaction Between CREPT/p15RS And RNA Polymerase II

CREPT and p15RS are two recently identified cancer-ralated genes thatshare high sequence similarites. CREPT is an oncogene with its coded proteinexpression-elevated in many tumor tissues. It promotes cell proliferation bypromoting transcription of cell cycle related genes including Cyclin D1. p15RScoded protein is related to p15INK4b, and its high expression inhibits cellproliferation. It’s a tumor suppressor gene. Both CREPT and p15RS consist ofan N-terminal RPR domain and a C-terminal domain, and the RPR domain couldinteract with RNA polymeras II C-terminal domain (CTD). While themechanism of function differences between CREPT and p15RS is not clear atpresent, this project aims to study the interaction between CREPT/p15RS andRNA polymerase II to provide structural and biochemical basis ofCREPT/p15RS functions.First, we expressed full-length CREPT and p15RS in E.coli, finding thatboth proteins are easily degraded.Then we did some structural and biochemical assay with CREPT-RPR andp15RS-RPR domains. Through fluorescence polarization analysis, we show thatCREPT-RPR and p15RS-RPR bind to different Pol II CTD phosphoisoformswith similar affinity and specificity. We also determined the crystal structure ofp15RS-RPR, which shows an eight-helix bundle. Sequence and structuralcomparisons with RPR domain of RTT103, a homolog of CREPT and p15RS inyeast, show that CREPT-RPR and p15RS-RPR are very similar withRTT103-RPR, revealing a structural basis for the similar binding profile ofCREPT-RPR and p15RS-RPR with RNA Polymerase II CTD.We also determined the crystal structure of CREPT-CTD. CREPT-CTD is along rod-like coiled-coil dimer, with each monomer consisting of four helixes.There are strong hydrophobic and electrical interactions between twoCREPT-CTD monomers. Through biochemical assays, we found thatCREPT-CTD and p15RS-CTD are both dimers in solution. We propose thatdimerization through the C-terminal domain enhances the binding strengthbetween CREPT or p15RS with RNA Polymerase II by increasing binding avidity.Our results collectively reveal the respective roles of N-terminal RPRdomain and C-terminal domain of CREPT and p15RS in recognizing RNAPolymerase II, and provide some structural and biochemical basis for functionof CREPT and p15RS.