Single-molecule analysis of transcription activation: dynamics of SAGA coactivator recruitment

Abstract

Transcription activators are said to stimulate gene expression by ‘recruiting’ coactivators, yet this vague term fits multiple kinetic models. To directly analyze the dynamics of activator–coactivator interactions, single-molecule microscopy was used to image promoter DNA, a transcription activator and the Spt–Ada–Gcn5 acetyltransferase (SAGA) complex within yeast nuclear extract. SAGA readily but transiently binds nucleosome-free DNA without an activator, while chromatin association occurs primarily when an activator is present. On both templates, an activator increases SAGA association rates by an order of magnitude and dramatically extends occupancy time. These effects reflect sustained interactions with the transactivation domain, as VP16 or Rap1 activation domains produce different SAGA dynamics. SAGA preferentially associates with templates carrying more than one activator. Unexpectedly, SAGA binding is substantially improved by nucleoside triphosphates but not histone H3 or H4 tail tetra-acetylations. Thus, we observe two modes of SAGA–template interaction: short-lived activator-independent binding to non-nucleosomal DNA and tethering to promoter-bound transcription activators for up to several minutes.