Abstract
The general transcription factor IID (TFIID) is the first component of the preinitiation complex (PIC) to bind the core promoter of RNA polymerase II transcribed genes. Despite its critical role in protein-encoded gene expression, how TFIID engages promoter DNA remains elusive. We have previously revealed a winged-helix DNA-binding domain in the N-terminal region of the largest TFIID subunit, TAF1. Here, we report the identification of a second DNA-binding module in the C-terminal half of human TAF1, which is encoded by a previously uncharacterized conserved zinc knuckle domain. We show that the TAF1 zinc knuckle aids in the recruit of TFIID to endogenous promoters vital for cellular proliferation. Mutation of the TAF1 zinc knuckle with defects in DNA binding compromises promoter occupancy of TFIID, which leads to a decrease in transcription and cell viability. Together, our studies provide a foundation to understand how TAF1 plays a central role in TFIID promoter binding and regulation of transcription initiation.
Highlights
transcription factor IID (TFIID) is a multi-subunit complex comprised of the TATA binding protein (TBP) and 14 TBP associated factors (TAFs) in humans[5]
A sequence alignment performed on full-length TAF1 using eight species that span the eukaryotic kingdom revealed two domains characterized by strictly conserved residues, which are absent in other regions of the protein (Fig. 1A)
The zinc knuckle (ZnK) found in TAF1 has a unique spacing shared by only a small number of annotated ZnK proteins, I factor and FAM90a, both of which have been annotated as interacting with DNA31,32
Summary
TAF1 contains an evolutionarily conserved zinc knuckle motif. TAF1 is an essential protein found in all eukaryotic organisms. These outward facing positively charged residues produce an interface to presumably interact with negatively charged nucleic acids Taken together, these features signify TAF1 ZnK has the potential to function as a DNA binding domain and contribute to TFIID promoter recognition. We observed reduced stimulation of cyclin D1 transcription by the TAF1 domain mutants compared to WT TAF1 (Supplemental Figure S1) These data suggest ZnK and WH are critical to recruit TFIID to core promoters and transcriptional initiation. To further define the TAF1 ZnK domain, we analyzed ZnA sequence with EVfold, a program that mines evolutionary information to detect connections between residues in a protein and predicts a three-dimensional shape based on the co-conservation of amino acids[42]. The identification of an additional DNA binding module in human TAF1 implies TFIID employs a multi-level approach to engage DNA, and with this knowledge we will continue to progress our understanding of promoter recognition and transcriptional initiation
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