Transient pausing by RNA polymerase II

Significance Transcription by RNA Polymerase II (Pol II) is a highly dynamic process that is tightly regulated at each step of the transcription cycle. We generated GFP-RPB1 knockin cells and developed photobleaching of endogenous Pol II combined with computational modeling to study the in vivo dynamics of Pol II in real time. This approach allowed us to dissect promoter-paused Pol II from initiating and elongating Pol II and showed that initiation and promoter proximal pausing are surprisingly dynamic events, due to premature termination of Pol II. Our study provides new insights into Pol II dynamics and suggests that the iterative release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.
Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell–imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.