Sequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The carboxy-terminal domain (CTD) of RNA Polymerase II is intrinsically disordered and crucial for transcription.
• CTD promotes liquid-liquid phase-separation (LLPS) of RNA Polymerase II in vivo.
• Direct biochemical characterization of CTD's role in LLPS is limited.

Purpose of the Study:

• To elucidate the sequence-encoded molecular grammar governing human CTD LLPS.
• To biochemically characterize the role of conserved heptad residues in CTD LLPS.
• To understand the interplay between CTD structure, LLPS, and phosphorylation by CDK7.

Main Methods:

• Systematic generation of CTD variants.
• In vitro biochemical assays.
• Molecular dynamics simulations.

Main Results:

• Tyrosine aromaticity and proline cis-trans isomerization are critical for CTD phase-separation.
• Cis proline conformation and SPXX motif β-turns promote a compact CTD ensemble and inter-residue interactions.
• Proline and tyrosine residues are essential for CTD phosphorylation by CDK7.
• CDK7 accelerates CTD phosphorylation within LLPS droplets, leading to hyperphosphorylation and release.

Conclusions:

• Conformationally restricted structures within CTD spacer regions, separating tyrosine residues, are vital for LLPS.
• The study reveals the molecular basis of CTD LLPS and its regulation.
• Findings provide insights into the dynamic control of transcription through CTD phase separation and phosphorylation.