Understanding Transcription through Structural Characterization of the CBP-p53 Transcriptional Coactivator-Activator Complex

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The process of transcription is essential to all life, controlling critical physiological processes such as cell growth, maintenance, development, and differentiation. With such a widespread influence on basic cellular processes, regulation of transcription is a high priority, with dysregulation of transcription being a major contributor to several diseases, including cancer, cardiovascular defects, and neurological disorders. While the process of the transcription “reaction” per se is quite simple, requiring essentially only an RNA polymerase enzyme, a DNA template and the appropriate nucleotide triphosphates, in cells transcription is much more intricate. Transcription relies on a specialized assembly of a host of dynamic and transient protein -protein interactions (PPIs) acting in a highly coordinated and regulated manner. At the crux of this assembly are transcriptional activators and coactivators. Remarkably, a given transcriptional activator or coactivator will use the same binding surface to complex with a myriad of binding partners including other transcription factors, coactivators, members of the transcriptional machinery and/or chromatin remodeling complexes. Thus, a central question that arises is what are the molecular recognition principles and mechanisms that govern the assembly of a given complex and how is selectivity in binding achieved for a given functional outcome. A clue to this query may reside in the presence of the intrinsically disordered regions (IDRS) within one or both binding partners present within these kinds of PPIS. For instance, in a given activator, disordered regions within their transcriptional activator domain (TAD) can adopt a distinct tertiary structure to suit the topology of the specific activator binding domain of a coactivator. On the flip side, a given coactivator activator binding domain (ABD) can undergo conformational remodeling fine tuning its docking surface to accommodate different protein binding partners. This structural adaptability and plasticity enables both specificity and promiscuously whilst at the same time bestowing seemingly unlimited regulatory potential of biological outcomes within the cell. Focusing on the master coactivators, CBP and p300 and their complexes with the prototypical transcriptional activator p53 and using a combination of classical biochemical methodologies and single particle electron microscopy (EM), the goal of this dissertation work has been to test the long-held hypothesis that multi-domain coactivators such as CBP and p300 undergo large conformational changes upon binding to activators. In chapter two of this dissertation, I describe both the biochemical and biophysical characterization of the intact activator-coactivator complex formed between the coactivators CBP and p300 and the activator p53 and initial single particle EM studies that not only support the feasibility of studying this complex by cryo-EM but also reveal the general architecture of CBP and the accompanying architectural rearrangements that occur upon complex formation with p53. Structural studies on the CBP•(p53)4 complex are expanded upon in chapter three of this dissertation which focuses on the characterization of this complex at higher resolutions by cryo-EM. Taken together, these structural studies have allowed for testing of the outstanding hypothesis that binding at one or more ABDs within CBP or p300 elicits global architectural rearrangements and provides evidence challenging long held beliefs that activators are the predominant flexible partner in transcription complexes.