The Role of Transcription Factor - DNA Residence Time in Transcription

Abstract

Transcription is regulated by the interplay of many different factors in a sequence of intrinsically stochastic processes. For most genes, this leads to short periods of activity (called bursts) followed by quiescence. Binding of transcription factors (TFs), either activators or repressors, represents the first step of transcription initiation. Fluorescence microscopy revealed that most TFs bind dynamically to DNA while each possesses an unique residence time. How this DNA residence time influences the activity of the TF remains unclear. We probe the influence of TF DNA residence time on transcription by generating TFs that target a certain gene locus and only differ in their DNA residence times. Therefore, we designed series of transcription activator-like effectors (TALEs) based TFs with varying numbers of nucleotide-recognizing repeat domains targeting the promoter region of a reporter gene. We characterized the DNA residence times of our TALE-TFs by single molecule tracking experiments in living cells. To determine the transcriptional output and frequency of bursts, we use single molecule RNA fluorescence in situ hybridization (smRNA-FISH). For a repressing system, a factor of six in DNA residence time accounts for a decrease of 50% in transcript level, by down regulation of burst frequency at constant burst size. Similarly, different DNA binding domains differentially affect transcription output in an activating system. Together our experiments demonstrate that the DNA residence time of TFs is a regulatory factor of transcription.