Structural characterization of the RNA polymerase II CTD in different phosphorylation states.

Abstract

RNA polymerase II (Pol II) transcribes protein-coding genes and coordinates co-transcriptional processes such as mRNA maturation and histone modification. The intrinsically disordered C-terminal domain (CTD) of the largest subunit of Pol II is composed of dozens of repeats with the consensus sequence YSPTSPS and serves as a flexible binding scaffold for co-transcriptional regulatory proteins. During transcription, the CTD undergoes constant post-translational modifications. These changing modifications constitute the CTD code, which specifies the position of Pol II on a gene and recruits specific regulatory proteins. Two major CTD phosphorylation marks, pSer5 and pSer2, are characteristic of the early and the late stage of transcription respectively. To characterize structural properties of CTDs with different phosphorylation patterns, we generated different CTD variants using enzymatic approaches and probed their local and global structures with carbon direct-detect NMR and small-angle X-ray scattering (SAXS). Particularly, carbon direct-detect NMR provides higher spectral resolution for intrinsically disordered proteins and allows direct visualization of prolines, which constitute over 20% of the CTD. Together, our structural characterization of the Pol II CTD in different phosphorylation states provides insights for how phosphorylation regulates the ensemble-function relationship of intrinsically disordered proteins.