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 motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and 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, drastically accelerating phosphorylation and promoting 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.

CEITEC Central European Institute of Technology Masaryk University Brno Czechia

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czechia

Zobrazit více v PubMed

Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 Ångstrom resolution. PubMed DOI

Harlen, K. M. & Churchman, L. S. The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain. PubMed DOI

Yang, C. & Stiller, J. W. Evolutionary diversity and taxon-specific modifications of the RNA polymerase II C-terminal domain. PubMed DOI PMC

Eick, D. & Geyer, M. The RNA polymerase II carboxy-terminal domain (CTD) code. PubMed DOI

Buratowski, S. The CTD code. PubMed DOI

Buratowski, S. Progression through the RNA polymerase II CTD cycle. PubMed DOI PMC

Jasnovidova, O. & Stefl, R. The CTD code of RNA polymerase II: a structural view. PubMed DOI

Jeronimo, C., Bataille, A. R. & Robert, F. The writers, readers, and functions of the RNA polymerase II C-terminal domain code. PubMed DOI

Egloff, S. & Murphy, S. Cracking the RNA polymerase II CTD code. PubMed DOI

Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S. & Cramer, P. A structural perspective of CTD function. PubMed DOI

Jasnovidova, O., Krejcikova, M., Kubicek, K. & Stefl, R. Structural insight into recognition of phosphorylated threonine‐4 of RNA polymerase II C‐terminal domain by Rtt103p. PubMed DOI PMC

Jasnovidova, O. et al. Structure and dynamics of the RNAPII CTDsome with Rtt103. PubMed DOI PMC

Kubicek, K. et al. Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1. PubMed DOI PMC

Mayer, A. et al. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. PubMed DOI

Cho, E.-J., Kobor, M. S., Kim, M., Greenblatt, J. & Buratowski, S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. PubMed DOI PMC

McCracken, S. et al. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. PubMed DOI

Komarnitsky, P., Cho, E.-J. & Buratowski, S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. PubMed DOI PMC

Ho, C. K. & Shuman, S. Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the recruitment and allosteric activation of mammalian mRNA capping enzyme. PubMed DOI

Cho, E.-J., Takagi, T., Moore, C. R. & Buratowski, S. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. PubMed DOI PMC

Descostes, N. et al. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells. PubMed DOI PMC

Brandts, J. F., Halvorson, H. R. & Brennan, M. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. PubMed DOI

Werner-Allen, J. W. et al. cis-proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72. PubMed DOI PMC

Xiang, K. et al. Crystal structure of the human symplekin–Ssu72–CTD phosphopeptide complex. PubMed DOI PMC

Schutkowski, M. et al. Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition. PubMed DOI

Goethel, S. F. & Marahiel, M. A. Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. PubMed DOI PMC

Schmid, F. X. Prolyl isomerase: enzymatic catalysis of slow protein-folding reactions. PubMed DOI

Favretto, F. et al. Catalysis of proline isomerization and molecular chaperone activity in a tug-of-war. PubMed DOI PMC

Zhang, M. et al. Structural and kinetic analysis of prolyl-isomerization/phosphorylation cross-talk in the CTD code. PubMed DOI PMC

Hanes, S. D. Prolyl isomerases in gene transcription. PubMed DOI PMC

Lu, K. P., Finn, G., Lee, T. H. & Nicholson, L. K. Prolyl cis-trans isomerization as a molecular timer. PubMed DOI

Bataille, A. R. et al. A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes. PubMed DOI

Andreotti, A. H. Native state proline isomerization: an intrinsic molecular switch. PubMed DOI

Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. PubMed DOI

Cramer, P. Organization and regulation of gene transcription. PubMed DOI

Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. PubMed DOI PMC

Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. PubMed DOI PMC

Kwon, I. et al. Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains. PubMed DOI PMC

Guo, Y. E. et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. PubMed DOI PMC

Alberti, S. Phase separation in biology. PubMed DOI

Martin, E. W. et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. PubMed DOI PMC

Ginell, G. M. & Holehouse, A. S. An Introduction to the Stickers-and-Spacers Framework as Applied to Biomolecular Condensates. In: PubMed

Rubinstein, M. & Dobrynin, A. V. Solutions of associative polymers.

Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. PubMed DOI PMC

Harmon, T. S., Holehouse, A. S., Rosen, M. K. & Pappu, R. V. Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. PubMed DOI PMC

Rekhi, S. et al. Expanding the molecular language of protein liquid-liquid phase separation. PubMed DOI PMC

Levitt, M. Conformational preferences of amino acids in globular proteins. PubMed DOI

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. PubMed DOI PMC

Gallardo, R., Ranson, N. A. & Radford, S. E. Amyloid structures: much more than just a cross-β fold. PubMed DOI PMC

Nelson, R. et al. Structure of the cross-beta spine of amyloid-like fibrils. PubMed DOI PMC

Eberhardt, E. S., Panasik, N. & Raines, R. T. Inductive effects on the energetics of prolyl peptide bond isomerization: implications for collagen folding and stability. PubMed DOI PMC

Panasik, N., Eberhardt, E. S., Edison, A. S., Powel, D. R. & Raines, R. T. Inductive effects on the structure of proline residues. PubMed DOI

Holmgren, S. K., Taylor, K. M., Bretscher, L. E. & Raines, R. T. Code for collagen’s stability deciphered. PubMed DOI

Buechter, D. D. et al. Co-translational Incorporation of Trans-4-Hydroxyproline into Recombinant Proteins in Bacteria. PubMed DOI

Cook, P. R. The organization of replication and transcription. PubMed

Wang, P. & Heitman, J. The cyclophilins. PubMed DOI PMC

Song, F. et al. Cyclophilin A (CyPA) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity. PubMed DOI PMC

Verdecia, M. A., Bowman, M. E., Lu, K. P., Hunter, T. & Noel, J. P. Structural basis for phosphoserine-proline recognition by group IV WW domains. PubMed DOI

Wang, J. et al. Allosteric breakage of the hydrogen bond within the dual-histidine motif in the active site of human Pin1 PPIase. PubMed DOI

Behrsin, C. D. et al. Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution. PubMed DOI

Zhou, X. Z. et al. Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and Tau proteins. PubMed DOI

Song, B., Bomar, M. G., Kibler, P., Kodukula, K. & Galande, A. K. The serine-proline turn: a novel hydrogen-bonded template for designing peptidomimetics. PubMed DOI

Trevino, S. R., Schaefer, S., Scholtz, J. M. & Pace, C. N. Increasing protein conformational stability by optimizing β-turn sequence. PubMed DOI PMC

Düster, R. et al. Structural basis of Cdk7 activation by dual T-loop phosphorylation. PubMed DOI PMC

Bao, Z. Q., Jacobsen, D. M. & Young, M. A. Briefly bound to activate: transient binding of a second catalytic magnesium activates the structure and dynamics of CDK2 kinase for catalysis. PubMed DOI PMC

Kato, M. & McKnight, S. L. A solid-state conceptualization of information transfer from gene to message to protein. PubMed DOI

Akhtar, M. S. et al. TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. PubMed DOI PMC

Peeples, W. & Rosen, M. K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. PubMed DOI PMC

Mikhaleva, S. & Lemke, E. A. Beyond the transport function of import receptors: what’s All the FUS about? PubMed DOI PMC

O’Flynn, B. G. & Mittag, T. The role of liquid–liquid phase separation in regulating enzyme activity. PubMed DOI PMC

López-Palacios, T. P. & Andersen, J. L. Kinase regulation by liquid–liquid phase separation. PubMed DOI PMC

Han, T. W. et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. PubMed DOI

Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. PubMed DOI PMC

Theillet, F.-X. et al. The alphabet of intrinsic disorder. PubMed DOI PMC

Semenov, A. & Rubinstein, M. Thermoreversible gelation in solutions of associative polymers. 1. DOI

Lin, Y., Currie, S. L. & Rosen, M. K. Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs. PubMed DOI PMC

Frey, S., Richter, R. P. & Görlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. PubMed DOI

Dignon, G. L., Best, R. B. & Mittal, J. Biomolecular phase separation: from molecular driving forces to macroscopic properties. PubMed DOI PMC

Flores-Solis, D. et al. Driving forces behind phase separation of the carboxy-terminal domain of RNA polymerase II. PubMed DOI PMC

Bremer, A. et al. Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains. PubMed DOI PMC

An, Y., Bloom, J. W. G. & Wheeler, S. E. Quantifying the π-stacking interactions in nitroarene binding sites of proteins. PubMed DOI

Joseph, J. A. et al. Physics-driven coarse-grained model for biomolecular phase separation with near-quantitative accuracy. PubMed DOI PMC

Schuster, B. S. et al. Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior. PubMed DOI PMC

Rana, U. et al. Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility. PubMed DOI PMC

Welles, R. M. et al. Determinants that enable disordered protein assembly into discrete condensed phases. PubMed DOI PMC

Souza, P. C. T. et al. Martini 3: a general purpose force field for coarse-grained molecular dynamics. PubMed DOI PMC

Thomasen, F. E. et al. Rescaling protein-protein interactions improves Martini 3 for flexible proteins in solution. PubMed DOI PMC

Thomasen, F. E., Pesce, F., Roesgaard, M. A., Tesei, G. & Lindorff-Larsen, K. Improving Martini 3 for disordered and multidomain proteins. PubMed DOI

Zerze, G. H. Optimizing the Martini 3 force field reveals the effects of the intricate balance between protein–water interaction strength and salt concentration on biomolecular condensate formation. PubMed DOI

van Teijlingen, A., Smith, M. C. & Tuttle, T. Short peptide self-assembly in the martini coarse-grain force field family. PubMed DOI PMC

Sasselli, I. R. & Coluzza, I. Assessment of the MARTINI 3 performance for short peptide self-assembly. PubMed DOI PMC

Regy, R. M., Thompson, J., Kim, Y. C. & Mittal, J. Improved coarse‐grained model for studying sequence dependent phase separation of disordered proteins. PubMed DOI PMC

Tesei, G. & Lindorff-Larsen, K. Improved predictions of phase behaviour of intrinsically disordered proteins by tuning the interaction range. PubMed DOI PMC

Murray, K. A. et al. Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies. PubMed DOI PMC

Ridgway, Z. et al. Analysis of proline substitutions reveals the plasticity and sequence sensitivity of human IAPP amyloidogenicity and toxicity. PubMed DOI PMC

Theillet, F.-X. et al. The alphabet of intrinsic disorder: I. Act like a Pro: on the abundance and roles of proline residues in intrinsically disordered proteins. PubMed DOI PMC

Rousseau, F., Serrano, L. & Schymkowitz, J. W. H. How evolutionary pressure against protein aggregation shaped chaperone specificity. PubMed DOI

Zhao, G. et al. Peptidyl-prolyl isomerase Cyclophilin71 promotes SERRATE phase separation and miRNA processing in PubMed PMC

Babu, M., Favretto, F., Rankovic, M. & Zweckstetter, M. Peptidyl prolyl isomerase A modulates the liquid–liquid phase separation of proline-Rich IDPs. PubMed DOI PMC

Eichner, T., Kutter, S., Labeikovsky, W., Buosi, V. & Kern, D. Molecular mechanism of Pin1-Tau recognition and catalysis. PubMed DOI

Lu, H. et al. Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. PubMed DOI PMC

Corden, J. L. RNA polymerase II C-terminal domain: tethering transcription to transcript and template. PubMed DOI PMC

Kornberg, R. D. Mediator and the mechanism of transcriptional activation. PubMed DOI

Jonkers, I. & Lis, J. T. Getting up to speed with transcription elongation by RNA polymerase II. PubMed DOI PMC

Core, L. & Adelman, K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. PubMed DOI PMC

Kwak, H. & Lis, J. T. Control of transcriptional elongation. PubMed DOI PMC

Zhou, Q., Li, T. & Price, D. H. RNA polymerase II elongation control. PubMed DOI PMC

Palacio, M. & Taatjes, D. J. Merging established mechanisms with new insights: condensates, hubs, and the regulation of RNA polymerase II transcription. PubMed DOI PMC

Stortz, M., Presman, D. M. & Levi, V. Transcriptional condensates: a blessing or a curse for gene regulation? PubMed DOI PMC

Richter, W. F., Nayak, S., Iwasa, J. & Taatjes, D. J. The mediator complex as a master regulator of transcription by RNA polymerase II. PubMed DOI PMC

Castellana, M. et al. Enzyme clustering accelerates processing of intermediates through metabolic channeling. PubMed DOI PMC

Sang, D. et al. Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding. PubMed DOI PMC

Gradia, S. D. et al. MacroBac: new technologies for robust and efficient large-scale production of recombinant multi-protein complexes. PubMed DOI PMC

Shis, D. L. & Bennett, M. R. Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. PubMed DOI PMC

Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. PubMed DOI PMC

Lamprecht, M. R., Sabatini, D. M. & Carpenter, A. E. CellProfiler PubMed DOI

Otsu, N. A. Threshold selection method from gray-level histograms.

R Core Team (2021): A language and environment for statistical computing. Vienna, Austria. https://posit.co/.

Team, Rs. RStudio: Integrated Development Environment for R (2022). https://posit.co/.

Wickham, H. et al. Welcome to the tidyverse. DOI

Wickham, H. ggplot2, Elegant Graphics for Data Analysis. (Springer-Verlag New York, 2016)

Clarke, E., Sherrill-Mix, S. & Dawson, C. Package ‘ggbeeswarm (2017). https://CRAN.R-project.org/package=ggbeeswarm.

Wilke, C. O. Tools for visualizing uncertainty with ggplot2 (2021). https://github.com/wilkelab/ungeviz.

Kassambara, A. ggpubr: ‘ggplot2’ Based Publication Ready Plots. https://rpkgs.datanovia.com/ggpubr/.

Welch, B. L. The generalization of ‘Student’s’ problem when several different population variances are involved. PubMed

Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. PubMed DOI PMC

Linhartova, K. & Falginella, F. L. Raw data and MD simulations files for the paper: “Sequence and Structural Determinants of RNAPII CTD Phase-separation and Phosphorylation by CDK7”. 10.5281/zenodo.10696484 (2024). PubMed

Zeiss Microscopy GmbH, C.

The PyMOL Molecular Graphics System. Version 2.0. Schrödinger, LLC.

Case, D. A. et al. AmberTools. PubMed DOI PMC

Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. DOI

Tribello, G. A., Bonomi, M., Branduardi, D., Camilloni, C. & Bussi, G. PLUMED 2: new feathers for an old bird. DOI

Kroon, P. C. et al. Martinize2 and Vermouth: unified framework for topology generation.

Dignon, G. L., Zheng, W., Best, R. B., Kim, Y. C. & Mittal, J. Relation between single-molecule properties and phase behavior of intrinsically disordered proteins. PubMed DOI PMC

de Jong, D. H., Baoukina, S., Ingólfsson, H. I. & Marrink, S. J. Martini straight: boosting performance using a shorter cutoff and GPUs. DOI

Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. DOI

Shabane, P. S., Izadi, S. & Onufriev, A. V. General purpose water model can improve atomistic simulations of intrinsically disordered proteins. PubMed DOI

Hornak, V. et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters. PubMed DOI PMC

Izadi, S., Anandakrishnan, R. & Onufriev, A. V. Building water models: a different approach. PubMed DOI PMC

Homeyer, N., Horn, A. H. C., Lanig, H. & Sticht, H. AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine. PubMed DOI

Park, S., Radmer, R. J., Klein, T. E. & Pande, V. S. A new set of molecular mechanics parameters for hydroxyproline and its use in molecular dynamics simulations of collagen‐like peptides. PubMed DOI

Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. PubMed

Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. DOI

Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An DOI

Hess, B. P-LINCS: a parallel linear constraint solver for molecular simulation. PubMed DOI

Michaud‐Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. PubMed DOI PMC

Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. PubMed DOI

Mao, A. H., Crick, S. L., Vitalis, A., Chicoine, C. L. & Pappu, R. V. Net charge per residue modulates conformational ensembles of intrinsically disordered proteins. PubMed DOI PMC

Flory, P. J. The configuration of real polymer chains. DOI

Dima, R. I. & Thirumalai, D. Asymmetry in the shapes of folded and denatured states of proteins. DOI

Shapovalov, M., Vucetic, S. & Dunbrack, R. L. A new clustering and nomenclature for beta turns derived from high-resolution protein structures. PubMed DOI PMC

Smith, P., Ziolek, R. M., Gazzarrini, E., Owen, D. M. & Lorenz, C. D. On the interaction of hyaluronic acid with synovial fluid lipid membranes. PubMed DOI

Distinct mechanisms of recognition of phosphorylated RNAPII C-terminal domain by BRCT repeats of the BRCA1-BARD1 complex

Off-pore Nup98 condensates mobilize heterochromatic breaks and exclude Rad51

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