Soegaard, TMM; (2006) The RNA polymerase II C-terminal domain: its phosphorylation and interaction with the mediator complex. Doctoral thesis , UCL (University College London).

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Abstract

The submitted thesis describes a biochemical investigation within the field of gene transcription. In yeast (Saccharomyces cerevisiae ) and the metazoans, the conserved RNA polymerase II (RNAPTJ) protein complex carries out regulated transcription. The C-terminal domain (CTD) of Rpbl (the largest subunit of RNAPII) consists of 26 hepta-peptide (YSPTSPS) repeats that are targeted by specific CTD-kinases and - phosphatases to change the phosphorylation of Ser2 and Ser5 resulting in two electrophoretically different forms of RNAPII - a fast migrating hypo-phosphorylated form and a slower migrating hyper-phosphorylated form. The changing patterns of phosphorylation modulate the interaction of multiple factors involved in transcription initiation, RNA processing, transcription elongation and RNAPII ubiquitylation throughout the transcription cycle. Prior to transcription a preinitiation complex (PIC) is assembled on the promoters of regulated genes. A plethora of regulatory pathways converge on a protein complex named Mediator - a central, functionally conserved protein complex that incorporates into the PIC to physically interact with RNAPII and which can only interact with hypo-phosphorylated RNAPII. Mediator plays the crucial role of relaying repressive and activating signals from transcription factors bound on the promoter to RNAPII poised at the beginning of the coding sequence of the gene. As Mediator is only found associated with promoter regions on chromatin RNAPII must dissociate from Mediator when transcription initiates and promoter escape occurs. The mechanism leading to dissociation of RNAPII from Mediator is unknown. A large conformational change in Mediator accompanies the association with RNAPII and the interaction is dependent both on the C-terminal domain of Rpbl (CTD) and of other members of the RNAPII complex. If hypo-phosphorylation is needed for Mediator to interact with RNAPII does hyper-phosphorylation lead to the dissociation of the two complexes Are other factors needed to bring about the dissociation of the holopolymerase complex By using purified forms of Mediator and holopolymerase and purified specific CTD kinases the work presented addresses these questions in vitro.

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