Widespread Backtracking by RNA Pol II Is a Major Effector of Gene Activation, 5′ Pause Release, Termination, and Transcription Elongation Rate

In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3′ flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5′ pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions. [Display omitted] •Dominant-negative TFIIS stabilizes backtracked pol II throughout human genes•RNA cleavage by pol II is essential for rapid activation of stress-inducible genes•Rescue from backtracking is important for escape from promoter-proximal pause sites•Rescue from backtracking is a major stimulus of rapid transcriptional elongation RNA polymerase II can backtrack and resume transcription after cleavage of the nascent RNA transcript. Sheridan et al. show that in vivo human pol II backtracks frequently and that RNA cleavage enhances gene activation, 5′ pause release, normal termination, and rapid transcription elongation.