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. 2000 Sep 12;97(20):10745–10750. doi: 10.1073/pnas.200365097

Figure 2.

Figure 2

Transcription of negatively supercoiled plasmids containing the CUP1 promoter by Pol II. (A) Map of the insert in pCUP1C-less(410). 3′URF, sequence from the termination region of the gene neighboring CUP1 in the yeast genome; UAS, upstream activating sequences (binding sites for Ace1 and HSF); T, putative TATA boxes; IE, initiation element (contains two point mutations as described in the text); synthetic cassette, synthetic C-less cassette. (B) Analysis of supercoiled pCUP1C-less(410) preparations in two-dimensional chloroquine gels. (Right) A mixture of plasmids prepared at superhelix densities: 0, −0.08, −0.12, and −0.16 (corresponding to 0, 26, 40, and 53 negative supercoils in a plasmid of 3474 bp). (Left) As the right panel but with plasmid prepared at native superhelix density (about −0.055) also added. (C) Transcription of negatively supercoiled pCUP1C-less(410) by purified Pol II. Analysis of radiolabeled transcripts in a sequencing gel. -C, -G: transcripts synthesized in the absence of CTP or GTP, respectively. Markers: pBR322 DNA digested with MspI. Superhelix densities (σ) are indicated. (D) Plot of amount of transcript initiated at the CUP1 promoter (phosphorimager units) against the square of the superhelix density for the data in C.