Turowski, T. W.; Petfalski, E.; Winz, M.-L.; Tollervey, D.

Transcription elongation is stochastic and driven by a Brownian ratchet mechanism, making it subject to changes in velocity. However, on regions occupied by multiple polymerases, notably the rDNA, DNA rotation plus torsion constrain polymerase molecules to proceed at the same rate generating \"torsional entrainment\". We report that release of entrainment, by co-transcriptional 3\'- end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5\'-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities were reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are targeted by both the exosome cofactor TRAMP and Rat1, promoting termination. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Biophysical modeling supported substantial (~10%) premature termination.