Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene

Transcription is a vital process activated by transcription factor (TF)
binding. The active gene releases a burst of transcripts before turning
inactive again. While the basic course of transcription is well
understood, it is unclear how binding of a TF affects the frequency,
duration and size of a transcriptional burst. We systematically varied the
residence time and concentration of a synthetic TF and characterized the
transcription of a synthetic reporter gene by combining single molecule
imaging, single molecule RNA-FISH, live transcript visualisation and
analysis with a novel algorithm, Burst Inference from mRNA Distributions
(BIRD). For this well-defined system, we found that TF binding solely
affected burst frequency and variations in TF residence time had a
stronger influence than variations in concentration. This enabled us to
device a model of gene transcription, in which TF binding triggers
multiple successive steps before the gene transits to the active state and
actual mRNA synthesis is decoupled from TF presence. We quantified all
transition times of the TF and the gene, including the TF search time and
the delay between TF binding and the onset of transcription. Our
quantitative measurements and analysis revealed detailed kinetic insight,
which may serve as basis for a bottom-up understanding of gene regulation.