TPL requires components of the nucleation step of the transcriptional pre-initiation complex (PIC) for repression. (A) Schematic of the steps of the transcriptional preinitiation complex formation on a promoter, accompanied by a cartoon representation of TFIID and TFIIA components. (B) Crystal structure of human TFIID PDB 6MZL (Patel et al., 2018). Both A and B were colored to represent which genes were identified by proximity labeling (red) and genetic interaction (blue) or both (purple). (C) Cytoplasmic split-ubiquitin system (CytoSUS) assays with candidate interacting proteins. Nub-3xHA is the N-terminal fragment of ubiquitin expressed with no fusion protein and is used as a negative control. −WL, −ADE: dropout lacking Trp, Leu, and Ade (growth control); −WLMH, −ADE: dropout lacking Trp, Leu, His, Met, and Ade (selective media). (D) Time-course flow cytometry analysis of SPARC^H1–H5 in selected anchor-away strains that include selected GTF components (Petrenko et al., 2017). (E) Time-course flow cytometry analysis of SPARC^H1–H5 in a Taf5 anchor-away strain (Taf5-2xFRB). (F) Heat map of all TFIID components in TS screens. Columns 1–6 are z-scores for all mutants that were recovered from all TS replicate screens. NAA – auxin application. (G) Top – gene map of the ScTaf5 protein. Middle – helical wheel diagram of the ScTaf5 LisH Helix 1 alpha helix. Bottom – protein alignment of ScTaf5 LisH Helix 1 domain and selected mutations. (H) Steady-state flow cytometry of taf5-15 with selected rescue constructs grown at the non-permissive temperature (30°C). Gray bar indicates the fluorescence of the wild-type rescue construct. (I) AID-Taf5 schematics and rescue experiments. Fluorescence were quantified by cytometry at 6 h after application of 5 μM IAA. (J) Cartoon schematic of the dCAS9-repressor experiment to test LisH repression function in HL-60 cell culture. (K) Quantification of CD4 protein levels by flow cytometry. Isotype control is provided to highlight the baseline fluorescence levels.