S CEREVISIAE GENERAL TRANSCRIPTION FACTOR TFIID

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transcription factor TFIID is a multi-subunit complex responsible for targeting and nucleating the assembly of RNA polymerase II and other associated factors to promoter start sites in eukaryotic organisms. It is important for the regulation of gene expression. Mis-regulation of gene expression can cause developmental defects, metabolic disorders, and cancer. Understanding the mechanism of transcriptional initiation is an important goal, and structural information about TFIID would contribute towards this end. The TFIID complex is estimated to contain 1.1-1.2 MDa of protein contributed from 15 individual subunits. Structural information for this large complex has been difficult to obtain due to expression and solubility problems for the isolated subunits and difficulty in obtaining sufficient material. Our goal has been to extend on negative stain electron microscopy studies of the yeast TFIID complex carried out by the group of P. Schultz using Tandem Affinity Purification (TAP-tag) isolated material. We will obtain cryo-EM reconstructions of the TFIID complex. This data will be used in our efforts to obtaining a high-resolution X-ray crystal structure. It would be of particular interest to further characterize the interactions of TFIID with other known basal factors using cryo-EM to further reveal the spatial relationships between the various components of the RNA Polymerase II transcription system at promoters.