Fig. 3: RNA–DNA hybrid formation in front of Pol II.
From: RNA polymerase II is a polar roadblock to a progressing DNA fork
a Outline of the experimental steps to backtrack Pol II and determine if RNA–DNA hybrid formation occurred. b Representative force-extension trace of the DNA fork interaction with an elongating Pol II in the head-on orientation, with minimal backtracking during the force clamp step. The DNA can be rezipped at step 3. c Representative force-extension trace of the DNA fork interaction with an elongating Pol II in the head-on orientation, with extensive backtracking during the force clamp step. The DNA cannot be rezipped at step 3. d Cartoon depicting the proposed fork and Pol II conformation after Pol II is backtracked upon head-on collision with the fork. The 3’ RNA extruded from a backtracked Pol II can hybridize with the lagging strand in front of Pol II. For ease of strand tracking, dsDNA or RNA–DNA hybrids are shown as two parallel strands, except for the inset where their helical structures are correctly depicted. The inset illustrates how the RNA–DNA hybrid formation restricts Pol II rotation and DNA unwinding, topologically locking Pol II on the DNA. e The minimum force reached during the attempt to rezip step vs the amount of backtracking during the force clamp step, in the presence and absence of RNase T1. A low minimum force characterizes an inability to rezip. The black dashed line indicates the initial Pol II position before the force clamp. The backtracked distance in base pairs may be estimated using a conversion factor of 1.0–1.2 bp/nm. (−) RNase T1, N = 46; (+) RNase, T1 N = 16. f Representative force-extension trace of the DNA fork interaction with an elongating Pol II in the head-on orientation in the presence of RNase T1. The DNA can be rezipped at step 3. Source data are provided as a Source data file.
