Fig. 4. Stalled polymerase complexes are predominantly backtracked.

(A) The role of transcription cleavage factor IIS in rescuing arrested Pol II elongation complexes. Following transcriptional pausing (top panel) the polymerase can backtrack along DNA (middle panel), which dislodges the RNA 3’-end from the polymerase active site (shown as red dot) and blocks further transcription. IIS induces internal cleavage of nascent RNA (bottom panel), re-aligning the 3’-end with the active site, such that Pol II can resume transcription. (B) Depletion of IIS leads to an increase in RNA length within the promoter-proximal region. Shown is the difference between the normalized number of reads in IIS-depleted and mock-treated samples, binned in 5-nt windows. (C) IIS-depletion affects RNA 3’-end positions but not permanganate reactivity profiles. RNA 3’-ends from mock-treated samples are shown in orange and IIS-depleted cells in green. Brackets denote regions of permanganate reactivity.