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 (TFIIS DN) 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 TFIIS DN, 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. TFIIS DN 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.
Features Small RNA-Seq Library Prep Kit for Illumina