Abstract

DNA in cells is often topologically closed, and in some cases, tightly looped by proteins typically associated with transcriptional repression. During transcription initiation, RNA polymerase (RNAP) is challenged to open duplex DNA, wherein RNAP generates torque that consequently overtwists DNA flanking the melted promoter. As initiation proceeds, the transcription bubble expands as RNAP synthesizes downstream RNA while maintaining upstream DNA contacts. We hypothesized that the mechanical strain imparted to DNA during initiation would repress transcription from DNA templates that restrict the relief of torsional stress. To test our hypothesis, we constructed circular DNA templates that are 100 to 108 bp in size (i.e., each initially twisted to various degrees) and quantified transcription initiation by the bacteriophage T7 RNAP. We find that transcriptional repression during initiation is dependent on the sign and magnitude of initial twist within the DNA templates. Surprisingly, however, we observe that for the most overtwisted templates, repression is relieved at positions beyond the promoter that are dependent on the initial DNA twist. To interpret these results, we used elastic rod and molecular dynamics simulations to predict the structures of both the RNAP and the circular DNA template during initiation. Our modeling studies confirm that RNAP is capable of overtwisting the circular DNA template to the point of buckling from a planar into a supercoiled conformation. Further analysis reveals that initial DNA twist determines at what position along the template supercoiling will occur during initiation and that adopting a supercoiled structure substantially relieves the torque encountered by the RNAP. Our results demonstrate that repression of RNAP during initiation is determined not only by the initial mechanics of the DNA template, but also by the torque generated by RNAP itself.

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