A dynamic conformational landscape underlies TFIID's ability to interact with core promoter DNA

Proper gene regulation is a problem faced by all organisms, ranging from single-celled bacteria to multi-cellular mammals. In order to respond to the changing environment, thousands of genes across the genome must be controlled in a coordinated manner. The process of evolution has placed these gene regulatory networks under constant selective pressure, which has resulted in the intricate assembly of protein-protein, protein-RNA, and protein-DNA interactions necessary for accurate gene expression. Considering that the mis-regulation of genes results in human diseases ranging from autoimmune disorders to cancers, it has been a major focus of modern molecular biology to understand the molecular mechanisms that determine these complex patterns of gene expression. By advancing our understanding of these processes, we will illuminate both the process of evolution in addition to providing better tools to diagnose and treat human diseases./nIn order to study the molecular underpinnings of transcription regulation in multi-cellular organisms, we have focused our investigation on the key human transcription factor known as TFIID. As a 13-14 protein complex, TFIID serves as an important regulatory hub during the process of transcription initiation by simultaneously interacting with distal activators and repressors, promoter DNA elements, and the basal transcription machinery. TFIID integrates these signaling cues to initiate RNAPII loading at specific genes across the genome, thus ensuring the survival of the cell and organism as a whole. Considering that TFIID is conserved throughout eukaryotic life, understanding its ability to license RNAPII transcription stands to provide deep insight into gene regulation across many clades of life./nTo address the structural basis for TFIID's ability to communicate with upstream activators and promoter DNA elements, we used single particle electron microscopy to visualize human TFIID's interactions with TFIIA and promoter DNA. First, we discovered that TFIID co-exists in two predominant and distinct structural states differing by a 100 angstrom translocation of TFIID's major sub-domain, lobe A. This result was surprising because this dramatic domain reorganization has been overlooked for the past 10 years. The functionality of this rearrangement was probed by measuring the conformational partitioning of TFIID in the presence of TFIIA and promoter DNA. The activator TFIIA modulates the transition between these structural states, as the presence of TFIIA and promoter DNA facilitates the formation of a novel rearranged state of TFIID capable of promoter recognition and binding. DNA-labeling and footprinting, together with cryo-EM studies, mapped the locations of the TATA, Inr, MTE, and DPE promoter motifs within the TFIID-TFIIA-DNA structure./n/nThese structural studies have significantly advanced the field of transcription initiation by providing mechanistic and regulatory insight into eukaryotic transcription initiation. The structure of TFIID-TFIIA-SCP represents a conceptual framework for interpreting the past 30 years of biochemical experimentation with purified transcription factors, explaining mbox{TFIID's} extended footprint (~100 bps) on promoter DNA. Additionally, through localization of the Inr, MTE, and DPE promoter motifs, this structure represents an important milestone for understanding these highly utilized promoter DNA elements. Finally, by placing this structure within the context of the extensive rearrangement observed for TFIID, the existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.

Advisor: Nogales, Eva