Figure 2. RNAPI transcription pausing in the 3′ ETS.

(A) Green line, distribution of RNAPI CRAC reads around the 3′ end of the 35S pre-rRNA; yellow line, folding energy in a rolling window of 65 nt, offset by 15 nt (the region within the polymerase) behind the polymerase at each nucleotide position. Data are represented as median (n = 6).

(B) Distribution of RNAPI CRAC reads around the 3′ end of the 35S pre-rRNA. Rnt1 processing sites (B0-1 and B0-2) and the reported major terminator (T1) are indicated. Black line, distribution of 5′ ends of RNAPI CRAC reads around the 3′ end of the 35S pre-rRNA. The major peak corresponds precisely to the 3′ B0 cleavage site. Green line, distribution of RNAPI CRAC read 3′ ends. The peaks of the 5′ and 3′ end distributions are positioned 25 nt apart. A cartoon shows the two B0 sites that are cleaved together across a stem structure by the RNase III homolog Rnt1. Peaks corresponding to the major 5′ (black) and 3′ (green) peaks are labeled on the plot and in the cartoon with round markers. Data are represented as median (n = 6).

(C) Read length distribution plot for all reads mapped to RDN37-2 (all) and reads mapped at the major peak within the 3′ ETS (B0-2 + 25 peak), marked with green in (B).

(D) Cartoon showing a decreased RNAPI elongation rate as a result of Rnt1 cleavage. RNAPI is released from torsional entrainment on the DNA by Rnt1 cleavage (top). This facilitates rotation of RNAPI together with the DNA (center) and allows a decreased elongation rate (bottom).

(E) Mapping 5′ ends of RNAPI (Rpa190-HTP) CRAC data reveals that AID-Rnt1 depletion is associated with loss of B0-2 processing ends within the nascent RNA. This confirms that RNAPI-associated reads are co-transcriptionally cleaved by Rnt1. Data are represented as median (n = 3).

(F) Comparison of RNAPI (Rpa190-HTP) 3′ mapping before and after AID-Rnt1 degron depletion. Rnt1 depletion leads to loss of the RNAPI peak between B0-2 and T1 sites. Solid lines indicate the median of −IAA (green) and +IAA (black). Blue filling indicates significant differences between datasets, which do not overlap with q2-q3 of each dataset. Data are represented as median (n = 3).

(G) RNAPI (Rpa135-HTP) CRAC shows increased readthrough in a rpa12ΔC strain in which endonuclease cleavage of backtracked transcripts is abrogated. Data are represented as median (WT, n = 7; rpa12ΔC, n = 4).

(H) rpa12ΔC leads to less efficient transcription termination of RNAPI. The plot compares WT and rpa12ΔC distribution (top) with differences extracted and plotted separately (center) and cumulative sum plots plotted (bottom). In rpa12ΔC, the signal is elevated downstream of termination site T1. Data are represented as median (WT, n = 7; rpa12ΔC, n = 4).

(I) Boxplot showing a fraction of reads downstream of the RDN37-2 termination site for WT and rpa12ΔC strains (p < 0.001, two-sided Wilcoxon test).