Uncovering the conditions that dictate the liquid-liquid phase separation of the CTD of RNAPII.

Abstract

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 CTD in model organisms such as S. cerevisiae, S. pombe and C. albicans, is composed of ∼26 copies of consensus repeats (Y1S2P3T4S5P6S7). However, CTD sequences of fungi isolated from geographically distinct locations from temperate to polar regions, show high sequence diversity at positions 1, 4, and 7. The CTD is highly conserved at its Ser-Pro motifs, which are targets of phosphorylation and cis-trans prolyl isomerization by Ess1. Ess1, an essential peptidylprolyl isomerase in yeast, has a highly conserved evolutionary relationship with the CTD. Here, we found that different CTDs isolated from yeasts found in geographically distinct regions ranging from temperate to polar climates undergo LLPS, but do so differently. We hypothesize Ess1 and phosphorylation may modulate the phase behavior of the CTD. The objective of this study is to determine how linear amino acid sequence, phosphorylation, and cis-trans prolyl isomerization influences CTD phase behavior. To do so, we utilize a variety of biophysical characterization assays such as UV-Vis spectroscopy, DIC microscopy, and phase separation prediction algorithms. These results will elucidate what conditions promote the LLPS of the CTD and its orthologs, thereby setting the stage for further investigating the conditions that promote the formation of transcription factories.