biostudies-literatureLinhartova KEuropean Research CouncilGrantová Agentura České Republiky (Grant Agency of the Czech Republic)Ministerstvo Školství, Mládeže a Tělovýchovy (Ministry of Education, Youth and Sports)9163https://www.ebi.ac.uk/biostudies/studies/S-EPMC11502803biostudies-literatureUnknown15(1)The intrinsically disordered 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. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motiNature communicationsSequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7.PMC11502803CZ.02.01.01/00/22_008/000457521-24460S20-21581YLX22NPO5103101001470LM2023042649030Falginella FLVacha RSebesta MLinhartova KStefl RMatl MfalseSequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7.The intrinsically disordered 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. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX moti2024-01-01T00:00:00Z2024 Oct2026-06-02T03:44:14.765Z2025-04-04T12:01:13.635ZS-EPMC115028033944858010.1038/s41467-024-53305-2