Initiation of transcription by RNA polymerase II (Pol II) is the outcome of a number of sequential events beginning with the binding of activators to their binding sites, which will trigger the recruitment of coactivator complexes and general transcription factors (GTFs) at promoters to allow the loading of Pol II into the preinitiation complex. In this process, coactivators play multiple crucial roles. SAGA (Spt-Ada-Gcn5-Acetyltransferase) and the ATAC (Ada-Two-A-Containing) complexes are two functionally distinct, but related, coactivator complexes with several enzymatic activities. The inactivation of yeast SAGA induced a strong decrease of nascent transcription from all expressed genes demonstrating a general requirement of SAGA at all transcribed yeast genes1.
To better characterize the behavior of GTFs, Pol II, SAGA and ATAC in living human cells we analyzed the dynamic nature of GFP-tagged factors with fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) and fluorescence correlation spectroscopy (FCS). FRAP and FLIP measurements indicated that TFIIB, TFIID, ATAC and SAGA subunits are highly dynamic and exhibit only transient interactions with the chromatin with no detectable immobile fractions. FCS measurements indicated that TFIIB, TFIID, ATAC and SAGA have two distinct diffusing populations in the nucleus: “fast” (free complexes) and a “slow” population (chromatin interacting complexes). Inhibition of transcription and reduced levels of histone H3K4me3 decreased the “slow”, chromatin interacting, population of SAGA and ATAC, demonstrating that the equilibrium between free and chromatin interacting SAGA and ATAC complexes is regulated by the active transcription-dependent chromatin landscape2.
In addition, a novel method will be presented allowing measurements of dynamic changes in the distribution of endogenous transcription factors (Pol II), and specific modified histones (gH2AX), at the nanometre scale4.