Date of Award
Doctor of Philosophy (PhD)
Chemistry & Biochemistry
Dylan J. Taatjes
James A. Goodrich
Amy E. Palmer
Robin D. Dowell
Transcription is a key checkpoint in regulating cell processes. The activity of RNA Pol II is modulated through a variety of mechanisms at various stages of transcription, and the Mediator complex is a central player in carrying out these changes. Mediator is a 1.2 MDa complex composed of 30 subunits. A primary function of Mediator is to bridge the activation domain of a transcription factor bound at an enhancer with Pol II and the general transcription factors at a promoter. Mediator is therefore a direct conduit for transcription factors to activate their target genes.
Chapter 2 describes the design of a peptide that blocks p53 activation, presumably by occluding the transactivation domain from interacting with Mediator. The peptide is selective for p53 over transcription activated by other signaling pathways, and seems to function in cells as well as in an in vitro transcription assay. The work outlines a novel method for specifically blocking TF-Mediator interactions, which could be useful therapeutically as well as in the design of molecular probes to better understand mechanisms of TF activation.
In Chapter 3, the confirmation of a backtracked RNAP state in transcription initiation is discussed. This newly verified state likely contributes to initiation being a rate limiting step and has implications for gene regulation in bacteria during NTP imbalances. In addition, some initial experiments and future directions are described for the study of the backtracked state in RNA Pol II transcription.
Some additional topics are discussed in the appendices. Mediator has been found to interact with a type of non-coding RNA called activating RNA, and work has been done to characterize Mediator-RNA interactions in vitro and at the whole genome level. Attempts to perform crosslinking followed by mass spectrometry (CX-MS) on Mediator to create a subunit interaction map are also described. The work resulted in several simplified methods of purifying distinct Mediator complexes. Finally, the role of the TFIIH kinase CDK7 in directing epigenetic changes through phosphorylation of the CTD of Pol II is explained.
Allen, Benjamin Levi, "Disruption of P53 Activated Transcription by a Rationally Designed Peptide" (2017). Chemistry & Biochemistry Graduate Theses & Dissertations. 221.
Available for download on Wednesday, August 28, 2019