ABSTRACT The RNA polymerase I (Pol I) enzyme that synthesizes large rRNA precursors, exhibits high rate of pauses during elongation, indicative of a discontinuous process. We propose here that Premature Termination of Transcription (PTT) by Pol I is a critical regulatory step limiting rRNA production in vivo . The Pol I mutant, SuperPol (RPA135-F301S), produces 1.5-fold more rRNA than the wild type (WT). Combined CRAC and rRNA analysis link increased rRNA production in SuperPol to reduced PTT, resulting in shifting polymerase distribution toward the 3’ end of rDNA genes. In vitro , SuperPol shows defective nascent transcript cleavage. Notably, SuperPol is resistant to BMH-21, a drug impairing Pol I elongation and inducing proteasome-mediated degradation of Pol I subunits. Compared to WT, SuperPol maintains subunit stability and sustains high transcription levels upon BMH-21 treatment. These comparative results show that PTT is alleviated in SuperPol while it is stimulated by BMH-21 in WT Pol I.

Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription