Přehled o publikaci

The carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. RNAPII CTD is intrinsically disordered and has been shown to promote liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the precise role of the conserved heptad residues in LLPS have been hampered by the lack of direct characterization of the biochemical properties of the CTD. Here, we generated a systematic array of RNAPII CTD variants to unravel the sequence-encoded molecular grammar underlying LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of residue Y1 and the presence of prolines govern CTD phase separation. The cis conformation of prolines and beta turns in the SPXX motifs contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. Serine residues within the CTD consensus sequence influence droplet morphology and inhibit aggregation. We further demonstrate that both prolines and the tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, which drastically accelerates CTD phosphorylation and promotes the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.