Project Summary The intrinsically disordered carboxy terminal domain (CTD) of RNA polymerase II (RNAPII) may nucleate the formation of transcription factories through a mechanism of liquid-liquid phase separation (LLPS). Transcription factories are densely packed membraneless organelles (MLOs) that contain all the necessary components to perform the transcription cycle. The intrinsically disordered carboxy terminal domain (CTD) of RNA polymerase II (RNAPII) in model organisms such as S. cerevisiae, S. pombe and C. albicans, is composed of ~ 26 copies of consensus repeats (Y1S2P3T4S5P6S7). However, we have recently shown that CTD sequences of fungi isolated from geographically distinct locations from temperate to polar regions, show high sequence diversity at positions 1, 4, and 7 and undergo LLPS differently. The CTD is highly conserved at its Ser-Pro motifs where it is the target of phosphorylation and cis-trans prolyl isomerization by Ess1. Ess1, an essential PPIase in yeast (Pin1 in humans), has a highly conserved evolutionary relationship with the CTD. Ess1 and phosphorylation may modulate the phase separation behavior of the CTD, but the molecular mechanism remains elusive. This proposal has two specific aims to test my hypothesis. In Aim 1 we will elucidate the mechanism of CTD LLPS by generating wild-type CTD and functional unit mutants and subjecting them to a series of turbidity assays with and without the presence of Ess1 to elucidate differences in LLPS due to sequence contribution. LLPS will be observed by naked eye, differential interference (DIC) microscopy, dynamic light scattering (DLS) and UV-Vis spectroscopy. In Aim 2 we will determine how regulators of CTD function, phosphorylation and isomerization by Ess1, influence the LLPS of the CTD. To do so we will adopt the same approach as Aim 1 but phosphorylate CTD constructs and induce the LLPS of these constructs with and without the presence of Ess1. For both aims 1 and 2 we will investigate the atomic structure of the CTD both phosphorylated and non-phosphorylated and in complex with Ess1. We will utilize nuclear magnetic resonance spectroscopy (NMR) and specialized pulse sequences that enable the detection of single amino acid residues to determine the isomerization state of the CTD which will enable the elucidation of Ess1 involvement in LLPS. Upon completion of this study, our expected outcomes are elucidation of the mechanism by which LLPS of the CTD recruits Ess1 and how cis-trans isomerization influences LLPS. We expect to have a positive impact by providing a molecular basis for the role of LLPS in mediating the assembly of transcription factories by the CTD, thereby enabling additional pathways for therapeutically targeting transcription associated diseases.