Abstract: During transcript elongation in vitro, backtracking of RNA polymerase II (RNAPII) is a frequent occurrence that can lead to transcriptional arrest. The polymerase active site can cleave the transcript during such backtracking, allowing transcription to resume. Transcript cleavage is either stimulated by elongation factor TFIIS or occurs much more slowly in its absence. However, whether backtracking actually occurs in vivo, and whether transcript cleavage is important to escape it, has been unclear. Using a yeast TFIIS mutant that lacks transcript cleavage stimulatory activity and simultaneously inhibits unstimulated cleavage, we now provide evidence that escape from backtracking via transcript cleavage is essential for cell viability and efficient transcript elongation. Our results suggest that transcription problems leading to backtracking are frequent in vivo and that reactivation of backtracked RNAPII is crucial for transcription.CI - Copyright 2010 Elsevier Inc. All rights reserved.