Multiple mechanisms of termination modulate the dynamics of RNAPI transcription

• Published in: Cell reports (Cambridge) Vol. 44; no. 3; p. 115325
• Main Authors: Petfalski, Elisabeth, Winz, Marie-Luise, Grelewska-Nowotko, Katarzyna, Turowski, Tomasz W., Tollervey, David
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.03.2025
• Subjects: biophysical modeling; DNA torsion; Exoribonucleases - metabolism; exosome; ribosome synthesis; RNA polymerase; RNA Polymerase I - genetics; RNA Polymerase I - metabolism; RNA-protein interactions; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; topoisomerase; transcription; Transcription Termination, Genetic; Transcription, Genetic; UV crosslinking; yeast; ribosome synthesis; transcription; RNA-protein interactions; UV crosslinking; biophysical modeling; CP: Molecular biology; topoisomerase; RNA polymerase; exosome; DNA torsion; yeast
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2025.115325

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 the RNA polymerase I (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 the exosome cofactor Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP), respectively. 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. [Display omitted] •Nascent pre-rRNA 3′ cleavage promotes RNAPI deceleration and termination•RNAPI undergoes early start-site proximal termination at sites of polymerase pausing•Biophysical modeling indicates ∼10% early termination of RNAPI•Model supports roles of additional rDNA repeats in buffering pre-rRNA transcription Petfalski et al. reveal complexities in RNAPI termination. Torsion release, through nascent pre-rRNA cleavage by Rnt1, favors RNAPI pausing and torpedo termination by the exonuclease Rat1, aided by Nsi1 roadblocks and RNAPI-mediated endonuclease cleavage. Termination of prematurely stalled RNAPI is facilitated by TRAMP-mediated nascent transcript polyadenylation and a reverse torpedo mechanism.