RNA polymerase II initiation factors show different dynamic behaviour upon induced transcription in live cells

Transcription by RNA polymerase II (Pol II) requires the ordered action of general transcription factors (GTFs) forming the pre-initiation complex (PIC). How these events unfold kinetically remains unclear. Upon transcription activation, we observe coordinated recruitment of Pol II and GTFs together with chromatin decompaction. Pol II and its Ser5-phosphorylated form accumulate and persist at promoters, whereas TFIIB and TFIIF engage transiently, revealing distinct dynamic regimes. We find specific residence kinetics consistent with rapid exchange of GTFs and a more stable initiating Pol II population. A quantitative kinetic model recapitulates the temporal ordering and relative amplitudes of factor recruitment, predicts recruitment times, and links transient GTF engagement to sustained Pol II occupancy. Steady state measurements and transcription inhibition corroborate model predictions. These results uncover a dynamic hierarchy in human PIC assembly and establish a quantitative framework that connects factor exchange kinetics to the regulation of Pol II activity in living cells.