Transcription Factor Regulation of RNA polymerase's Torsional Capacity

During transcription, RNA polymerase (RNAP) supercoils DNA as it forward-translocates. Accumulation of this torsional stress in DNA can become a roadblock for an elongating RNAP and thus should be subject to regulation during transcription. Here, we investigate whether, and how, a transcription factor may regulate the torque generation capacity of RNAP and torque-induced RNAP stalling. Using a real-time assay based on an angular optical trap, we found that under a resisting torque, RNAP was highly prone to extensive backtracking. However, the presence of GreB, a transcription factor that facilitates the cleavage of the 3′ end of the extruded RNA transcript, greatly suppressed backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 to 18.5 pNnm. Analysis of the real-time trajectories of RNAP position at a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism that limits transcription against DNA supercoiling and the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings broaden the potential impact of transcription factors on RNAP functionality.