Pausing by E. Coli RNA Polymerase at LacI and EcoRI Protein Roadblocks

The synthesis of messenger ribonucleic acid (mRNA) from template deoxynucleic acid (DNA) by RNA polymerase (RNAP) decodes genetic information in all forms of life. In vitro, motor enzyme RNAP translocates at up to 20-25 base pairs per second, but forces and transcription factors can modulate activity and intrinsic pausing, critical for regulation. Previous studies suggest RNAP may backtrack after intrinsic pausing, which may prolong the inactive state, upon encountering a physical obstacle along the template DNA. It is uncertain whether RNAP actively disperses a roadblock or passively waits for it to dissociate. DNA binding proteins LacI and EcoRI were used as site-specific roadblocks, and E. Coli RNAP pause times were measured as a function of forces opposing or assisting RNAP translocation via magnetic tweezers. Pauses were also measured in the presence of GreA, a protein that rescues backtracked RNAPs by cleaving nascent RNA backed up into the catalytic site. Regardless of magnitude, forces opposing RNAP at LacI increased average pause durations compared to assisting forces. However, including GreA eliminated this difference. Moreover, the addition of GreA rescued complexes stalled indefinitely at EcoRI. Overall, backtracking by transcription elongation complexes extends pauses at EcoRI and LacI roadblocks unless GreA is present, but these two roadblocks must dissociate before RNAP can proceed. Our biomechanical measurements elucidate how forces on the genome affect RNAP behavior at roadblocks.