Fig. 4: TFIIS and RNase H facilitate RNA–DNA hybrid removal from backtracked Pol II.

a Outline of the experimental steps to backtrack Pol II, form RNA–DNA hybrid, and detect Pol II location after RNA–DNA hybridization. b Representative force-extension traces showing the interaction of the DNA fork with an elongating Pol II in the head-on orientation without TFIIS or RNase H (top plot), with TFIIS (middle plot), and with RNase H (bottom plot). For the trace without TFIIS or RNase H, after Pol II is backtracked in step 2, DNA cannot be rezipped in step 3. Pol II is further backtracked when checked during step 4. For the trace with TFIIS, after Pol II is backtracked in step 2, DNA cannot be rezipped in step 3. However, DNA becomes rezipped (indicating hybrid removal), and Pol II has forward translocated when checked during step 4. For the trace with RNase H, after Pol II is backtracked in step 2, DNA cannot be rezipped initially but becomes rezipped subsequently (indicating hybrid removal) during step 3. Pol II has also forward translocated when checked during step 4. c Percentage of traces that showed hybrid removal. The error bars represent SEMs of the counting errors (−TFIIS and −RNase H, with N = 26; +TFIIS, N = 33; +RNase H, N = 20). d Cartoons illustrating how TFIIS and RNase H can facilitate the release of the topologically locked Pol II by severing the 3’ RNA. Source data are provided as a Source data file.