Figure 4. Rat1 and TRAMP are found at sites of slowed RNAPI elongation.
(A) Rat1 associated with sites of slowed/paused RNAPI. The 5′ ends of Rat1-HTP mapped reads are presented. Data are represented as mean (n = 2).
(B) RNAPI (Rpa190-HTP) and Rat1 peak metaplots across the rDNA (RDN37). The +643 peak was excluded from this analysis (see also Figure S4B).
(C) Cumulative difference map for RNAPI (Rpa190-HTP) in rat1-1 versus WT. The rat1-1 mutation leads to rearrangements of RNAPI-associated peaks within the 5′ ETS region. The RNAPI peaks are less prominent and shifted in rat1-1 mutant when compared to WT cells.
(D) Fraction of oligo(A)+ reads recovered in RNAPI CRAC data (non-coded An ≥ 3), mapped across the 5′-ETS. Data are represented as median (n = 6).
(E) RNAPI CRAC peak metaplot for 5′ ETS, comparing the 3′ ends of the reads (green) with poly(A) reads (maroon). As expected, the Rpa190 distribution matches the metapeak plot giving a low signal.
(F) Schematic of the in vitro assay for transcription, backtracking, and adenylation.
(G) Purified Trf4-Air1/2 can add poly(A) tails to nascent RNA associated with backtracked RNAPI. Transcripts were internally radiolabeled and visualized with an image analyzer (Fuji-Film).
(H) Trf4 oligo-adenylates the 3′ end of backtracked, nascent RNA in vitro extruded from RNAPII. The assay was performed as outlined in (F) but using purified RNAPII and TFIIS in addition to Trf4-Air1/2.