Fig. 7
The Spt20 HIT region is conserved and sufficient to incorporate Tra1 into SAGA. a Schematic illustration of the different Spt20 truncation mutants constructed to identify the Head Interacting with Tra1 (HIT) region of Spt20 from S. cerevisiae. Distinct colours depict Spt20 domains, defined as Homology Boxes (HB) and a Low Complexity Region (LCR). Each mutant is named after to the last residue present in the truncation mutant, which shortens the LCR to various extent, as illustrated. b Silver staining of S. cerevisiae SAGA complexes purified from WT and spt20 truncation mutants, using Spt7 as the bait. Below is an anti-HA western blot of HA-Spt7-TAP in a fraction of the input used for TAP. Data are representative of three independent experiments. Hashtag indicates a doublet band likely resulting from an artefact of silver staining. In S. cerevisiae, the HIT region of Spt20 is narrowed down to residues 474–492 (purple-coloured box) and predicted to fold into a α-helix by PSIPRED70 (left). c LC-MS/MS analyses of S. cerevisiae SAGA complexes purified from WT, spt20–492, spt20–380, spt20–408, and spt20–474 truncation mutants, as in panel b. LFQ intensity ratios of Tra1 were normalised to the bait, Spt7. Ratios from two independent experiments are plotted individually with the mean (black bar). d Western blot analyses of V5-tagged Spt20 truncation mutants in SAGA complex eluates from panel b. e GST pull-down of S. pombe protein extracts from WT, tra1-Sptra2, and tra1-Sctra1 strains, using GST fused to S. pombe (Sp) Spt20 HIT region (residues 282–324) (top right panel) or GST fused to S. cerevisiae (Sc) Spt20 HIT region (residues 468–537) (bottom right panel). GST alone serves as a negative control for background binding to glutathione beads. Anti-FLAG western blotting is used to detect WT and hybrid Tra1 proteins bound to GST or GST-HIT columns (right panel) and in a fraction (0.6%) of the input used for the pull-downs. Data that are representative of three independent experiments. Coomassie blue staining of purified GST fusion proteins are shown on the right. Source data are provided as a Source Data file
