Evolution, structure and assembly of basal Transcription Factor II D

Abstract

Numerous transcription regulatory players have emerged in order to meet the increasing demand for transcriptional complexity in the eukaryotic branch of life. Being the first member of the pre-initiation complex to engage genes at their core promoters and accurately stabilize the RNA polymerase II (RNAPII) enzyme at the transcription start site (TSS), TFIID protein complex is key player in eukaryotic transcription initiation. The work described in this thesis examines comprehensively the evolutionary history of TFIID and places its origins in a common with the SAGA complex ancestor, dating prior to the last eukaryotic common ancestor (LECA). Through numerous duplication and sub-functionalization events, a gradual divergence of the two complexes is observed. Notably, while the core subunits of TFIID remain mostly conserved, dynamic gains and losses of epigenetic reader domains accompany the expansion of the eukaryotic branch, indicating continuous readjustment of the complex to the transcriptional demands and rules of different organisms. The structure of human TFIID is extensively analysed through a cell-based expression system coupled with affinity enrichment and label-free quantitative mass spectrometry analyses. Key mechanistic steps in the formation and dynamics of the complex were identified, including: 1) nuclear holo-TFIID assembly from discrete cytoplasmic submodules, 2) checkpoint role for the chaperonin complex, TRiC/CCT, during the formation of a central for TFIID integrity submodule, TAF5/TAF6/TAF9, 3) the atomic resolution of TAF5/TAF6/TAF9, revealing a novel TAF5/TAF9 interface with a crucial role in the release of newly-synthesized TAF5 from the TRiC/CCT, 4) disintegration of holo-TFIID once chromatin-bound to a primarily core-TFIID structure and a specific loss of TBP, as the protein engages other transcription players onto the DNA, and 5) a canonical holo-TFIID formation by its non-canonical TAF1L paralog, with integration efficiency dependent on the presence or absence of the canonical TAF1 subunit. Altogether, the work described in the thesis exemplifies comprehensive experimental and computational methods for the characterization of evolutionary and structural dynamics of large molecular machineries. Being central for the initiation of accurate eukaryotic transcription events, such an extensive picture of TFIID gives an opportunity to rationally place its structure and function in the context of the immense molecular network of eukaryotic transcription.