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

The synthesis of the large rRNA precursors is achieved by the RNA polymerase I (Pol I) enzyme. During elongation, Pol I exhibits a highly discontinuous process with a high rate of pauses. Here, we propose that Pol I Premature Termination of Transcription (PTT) is a major regulatory step limiting rRNA production in vivo. We previously isolated a mutant of Pol I carrying the RPA135-F301S mutation. This mutant (hereafter called SuperPol) is able to produce 1.5-fold more rRNA than wild-type (WT). Combining crosslinking and analysis of cDNAs (CRAC) and rRNA analysis we could show that increased rRNA production in SuperPol is associated with a reduced premature release of nascent transcripts and a shift of the polymerase distribution toward the 3' end of rDNA genes. In vitro, SuperPol shows a defect in nascent transcript cleavage combined with an increased ability to resume elongation after a pause, to the detriment of transcriptional fidelity. SuperPol is resistant to high concentrations of BMH-21, a drug that severely impairs Pol I elongation and triggers the proteasome-mediated degradation of the largest subunits of Pol I, in both human and yeast. Compared to WT, SuperPol retains subunits stability and maintains high levels of Pol I transcription upon BMH-21 treatment. These results show that PTT is both alleviated in SuperPol and targeted by BMH-21 in WT Pol I. Overall design: CRAC protocol performed on samples containing HTP-tagged proteins: Rpa135(WT)-HTP, Rpa135(F301S)-HTTP s were analyzed and in presence of BMH-21.