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Abstract

The TATA-box binding protein (TBP) is an evolutionarily conserved basal transcription factor (TF), and a common component of the pre-initiation complex (PIC) for all three eukaryotic RNA polymerases (Pols). While RNA Pol III remains universally TBP-dependent, RNA Pol I and II in mammals have evolved TBP-independent mechanisms, in contrast to strict TBP essentiality in yeast. We investigated cross-species TBP functionality and found that yeast TBP and murine TBP paralogs cannot fully replace endogenous TBP in mouse embryonic stem cells (mESCs), with rescue capacity correlating with RNA Pol III occupancy. Interestingly, yeast TBP can engage Pol I and II promoters in mESCs, indicating retained core promoter recognition. Conversely, mouse TBP and its core cannot fully complement TBP depletion in yeast, affecting growth and recruitment of RNA Pol II and III. Using single-molecule imaging, we show that DNA-binding dynamics are largely encoded by the conserved TBP core domain, while the intrinsically disordered N-terminal domain modulates residence time on DNA and transcriptional responses to stress. These findings reveal that structurally conserved and divergent TBP domains co-evolved to balance essential transcriptional functions with species-specific regulatory flexibility, highlighting mechanistic differences in transcription initiation across eukaryotes and the nuanced roles of TBP homologs in gene regulation.