Exploring the mechanism of RNA polymerase passage through a transcription factor barrier at the single-molecule level

Proper regulation of RNA transcription is critical for the homeostasis of all organisms. In contrast to the vast knowledge obtained on initiation phase, how transcription is regulated at the elongation phase is only beginning to be understood. In particular, as RNAP transcribes an RNA molecule, it encounters a multitude of DNA-bound complexes. As a result, RNAP may pause or backtrack. Of particular interest are TFs, which bind to promoters and enhancers in order to regulate initiation but can also recognize sequences inside gene bodies. How does RNAP cope with a bound TFs, and whether they can regulate elongation, are still open questions. Using optical tweezers and the Early growth response protein1 (egr-1) as a model, we established that the passage time of RNAP is modulated by the presence and orientation of egr-1. Surprisingly, TFs can bind DNA inside the footprint of the RNAP, but its stability is compromised. This destabilization is not a monotonous function of the position of the TF, suggesting that interactions with specific elements in the complex and a heterogenic structure of RNAP are responsible for TF displacement. Additional experiments revealed that RNAP interactions with the DNA are not uniform but include two main regions of protein-DNA interactions nearly 45 bp away from the active site, significantly further than previously reported. Together, our data suggest that specific domains in the RNAP structure are responsible for modulating the TF residence time, thus providing a mechanism for its active and dynamic removal during RNAP elongation.