Figure 3.

A Pol I minigene to study transcription termination. (A) Top: Scheme of the ribosomal minigene, including Pol I promoter plus upstream intergenic sequence (gray), selection fragment derived from the human β-globin gene (black) and Pol I terminator fragment (white) including Rnt1 cleavage site (triangle), T1 and Reb1-binding site (oval). Sizes in base pairs are indicated below. A and B show position of the PCR products for the RT–qPCR analyses in Figure 4B–D. Bottom: schematic of the terminator mutants incorporated into the minigene. (B) Primer extension showing authentic Pol I 5′-ends are produced from the minigene. Arrow indicates a single primer extension product corresponding to correctly initiated Pol I transcripts. (C) TSS detection on the Pol I minigene by RT–qPCR. The analysis was conducted in WT (left) or rat1-1 sen1-1 (right) cells, transformed with the indicated constructs. Reverse transcription was primed with an oligo selective for the plasmid-encoded transcripts, PCR with a communal reverse primer and different forward primers to generate products 1, 2, 3 and 4, shown in the scheme below. PCR efficiency was normalized to T3 transcript produced in vitro (T3 promoter is indicated).