Multiple Mechanisms of Termination Modulate the Dynamics of RNAPI Transcription

Transcription elongation is stochastic, driven by a Brownian ratchet, making it subject to changes in velocity. On the rDNA, multiple polymerases are linked by “torsional entrainment”, generated by DNA rotation. 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 are 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 terminated by forward and reverse “torpedoes”; Rat1 and exosome cofactor TRAMP. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Mathematical modeling supported substantial premature termination. These basic insights into transcription in vivo, will be relevant to many systems. Overall design: samples were analysed in total from a strain carrying HTP tagged RNAPI and untagged strain as a negative control. Duplicate or multiplicate experiments were carried out.