RNA Pol III initiation and termination sites on coligo 15a-3. (A) Denaturing polyacrylamide gel showing in vitro Pol III transcripts (IVT) made from coligo 15a-3 and, for reference, coligo 122. The RNA within the dashed line rectangle was cut from the gel for coligo-seq cDNA library preparation. Spiral arrow indicates tandem multimer transcripts (n > 1) resulting from termination read through. The periodic transcript pattern indicates similar initiation and termination sites in n = 2,3,4 multimer transcripts. A line between lanes indicates lanes originally separated on the same gel. RNA was visualized by uniform [α-32P]-UTP incorporation; multimers are overrepresented by multiples of incorporated label. Phosphorimager quantitation is corrected for uniform labeling and rounded off. M, RNA marker. (B) Read length distribution of cDNA library after Illumina sequencing. (C) The 15a-3 Pol III transcription initiation (i.e., start) sites (Tss, green bars) and termination (Term.) sites (red bars) were compiled and mapped to the predicted 15a-3 secondary structure. Read Coverage indicates the percentage of the total reads summarized by the red and green bars. If no number is shown next to a nucleotide, then reads initiating or terminating there were <1% of the total reads. LL, larger loop; SL, smaller loop; Stem, double-stranded region. Black triangle, circligation site. Curved line, possible Pol III termination sequence. Transcription proceeds counterclockwise around the coligo as drawn. (D) Most abundant n = 1 transcript. ppp, 5′ triphosphate.
