Extended Data Fig. 2: Dynamics of Spt7, Gcn5, and activator binding.
From: Single-molecule analysis of transcription activation: dynamics of SAGA coactivator recruitment
a. Three representative time records of SAGA and activator fluorescence intensity on non-chromatinized templates in the presence of 10 nM Gal4-SNAPDY649-VP16. Periods of fluorescent protein colocalization with the DNA template are colored (Spt7-SNAPDY549 in green; activator Gal4-SNAPDY649-VP16 in red). b. Three representative time records of fluorescence intensity for Spt7-SNAPDY549 and Gcn5-DHFRCy5TMP (extract from strain YSB3670), imaged in the presence of saturating, unlabeled Gal4-VP16 activator. c. Cumulative time-to-initial-binding distributions for Spt7-SNAPDY549 (left panel, green) and Gcn5-DHFRCy5TMP (right panel, orange). Gray curves show corresponding background binding to off-target sites on slide surface. Dashed lines show fits to a single-exponential specific binding model, with association rates and active fraction (Af) listed. d. Dual-color rastergram of Spt7-SNAPDY549 and Gcn5-DHFRCy5TMP binding events on DNA template. Binding records from 100 randomly selected template locations were binary-coded (color indicates colocalization with DNA) and sorted by time to first Spt7 binding, from bottom to top. Binding of Spt7 (green), Gcn5 (red), or both (blue) are overlaid. e. Dual color rastergrams for Spt7-HALOJF646 (green) and TBP (Spt15-SNAPDY549, red), sorted by time of initial Spt7 binding. Two DNA templates were analyzed on the same slide, with the standard 5xGal4bs-CYC1 construct (Fig. 1b) in left panel and a 5xGal4bs-RPS5 construct in right panel. f. Probability density of Spt7 binding survival intervals fit to a bi-exponential decay model. Dark green points (± S.E.) show SAGA binding with 10 nM activator, light blue points with no activator. Red and pink lines show fits to a bi-exponential decay model for both. Numbers of observations for statistics and fit parameters are given in Supplementary Table 6.
