Extended Data Fig. 8. Rewinding of transcription bubble triggers RNA release.

a. The sequence of DNA templates used in Fig. 3h. b. The in vitro transcription assay suggests that dsDNA rewinding is required for intrinsic termination at the ϕt₅₀₀ terminator. Data are presented as mean ± SD, n=3 biologically independent experiments, ****P<0.0001; two-tailed unpair t-tests. Raw data for the gels can be found in Supplementary Fig. 1g. c. Mapping the hairpin stem distance from U8 that triggers termination at U7 or U8. A template encoding a variant of λt_R2 terminator lacking the upstream half of the terminator hairpin was used (from pRM1234, Supplementary Table 1). Core-RNAP was halted at G33, and backtracking was prevented by addition of asDNA (#14483). Transcription was restarted after adding different asRNAs and a master mix with (ATP + GTP + CTP to yield final concentrations of 150 μM, and UTP to 10 μM). Samples were collected and mixed with stop buffer after 10, 20, 30, and 60 sec. The remaining fraction was then chased by all 4 NTPs to a final concentration of 650 μM each. Termination starts taking place when the hairpin is −11 away from U8. Results from a single replicate are shown. Raw data for the gel can be found in Supplementary Fig. 1h.