The Pol III transcription machinery mediates genome-wide roadblock of Pol II. (A) Metagene analysis on 100 bp upstream of tRNA genes, shown with PATs accumulating after nuclear depletion of Rrp6p and Dis3p (black dotted line) and Pol II–associated nascent 3′-ends in WT (black solid line) and dst1Δ (gray solid line) strains. The DNase I–resistant signal (gray dashed line) shows the footprint of the Pol III transcription factor TFIIIB. (B) Genome-browser view for SNR53 showing PATs accumulating upon nuclear depletion of the indicated strains. Pol II occupancy is shown at the bottom plotted from the Rpb2 PAR-CLIP signal. (C) Northern blot analysis on SNR53 termination products with probes to the mature snR53 (left) or the proximal downstream region (middle). SNR53 3′-extended transcripts were analyzed after 1 h of rapamycin treatment of the indicated FRB-tagged strains (right). The right four lanes are the same samples as from the middle panel. Note the size reduction in the 3′-extended snR53 due to the deletion of the tRNA gene.