NMR spectroscopy of liquid-liquid phase separation related to transcription

by Irina Lushpinskaia
Doctoral thesis
Date of Examination:2023-08-28
Date of issue:2024-06-05
Advisor:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Markus Zweckstetter
Referee:Dr. Johannes Soeding
Persistent Address: http://dx.doi.org/10.53846/goediss-10514

 

 

Files in this item

Name:PhD-Thesis_Lushpinskaia_eDiss.pdf
Size:6.97Mb
Format:PDF
Description:Ph.D. Thesis

The following license files are associated with this item:

Abstract

English

The Carboxyl-Terminal Domain (CTD) of the largest subunit RPB1 of RNA Polymerase II (Pol II), is a disordered low-complexity region which is crucial for pre-mRNA transcription and processing. Due to its highly repetitive, intrinsically disordered sequence, the unphosphorylated CTD provides the clustering and phase separation of RNA Pol II. Clustering of this region through liquid-liquid phase separation (LLPS) is a comprehensive process that remains unclear at the atomic level. Here, we investigate the liquid-liquid phase separation (LLPS) properties of human and yeast CTDs. Phase separation of CTD occurs at physiological temperature in the absence of crowding agents and is reversed upon cooling, in agreement with a lower critical solution temperature (LCST). Point mutations of tyrosine residues and changing positions of proline residues within the CTD sequence demonstrate that a broad spectrum of interactions involving tyrosine residues imparts temperature- and concentration-dependent self-coacervation of the CTD. Aromatic interactions are critical for CTD phase separation and cannot be compensated by increase in hydrophobicity. By combining NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations, a high-resolution structural description of CTD is obtained. In addition, these results demonstrate the presence of diverse tyrosine engaging interactions, including tyrosine-proline contacts, in condensed and diluted states of CTD. These interactions are decisive to stabilize local structures of CTD and to regulate LLPS through CH/π-interactions. The contribution of tyrosine proline contacts for hCTD simulations are shown in comparison to the statistics obtained for other disordered LLPS proteins such as LGE1 and FUS, simulated using the same modeling framework. These results are compared with the recent Cryo-EM structure “TFIID-based PIC-Mediator holo-complex in fully-assembled state” (PDB 7ENC), where similar interactions were pointed out. Together with published data, this work provides understanding of the driving forces of CTD self-coacervation and therefore basis to better understand CTD mediated Pol II clustering in eukaryotic gene transcription
Keywords: CTD; RNA Polymerase II; Transcription; Liquid-liquid Phase Separation; Nuclear Magnetic Resonance Spectroscopy; Intrinsically Disordered Proteins/Regions
 
Statistik