Fig. 2.

Reconstituted transcription initiation system supports 5′ capping and reinitiation. (A) To confirm reinitiation from the G-less 49 promoter, Northern blot analysis was performed on G-less 49 transcripts that were generated in the presence or absence of Cet1-Ceg1 and Abd1. Probes antisense to nt 1 to 25 or nt 26 to 49 were used. Transcripts generated by the second round of transcription are indicated by a blue line. (B and C) Potassium permanganate (KMnO₄) footprinting assays with the G-less 49 (Upper) and the G-less 27 (Lower) templates (−122/+97) to detect single-stranded regions. The 5′ ends of the template (B) or nontemplate (C) strands were labeled with ³²P. After incubation of the initiation reactions for 20 min, 18 mM KMnO₄ was added. In C, an ∼50-bp region downstream of the TATA box is enlarged and shown with darker exposure (Lower). KMnO₄ reactive positions on template and nontemplate strands are indicated by orange and green dots, respectively. (D) G-less 49 (Top) and G-less 27 (Bottom) DNA sequences showing KMnO₄ reactive residues. The TATA box is shown in red, and TSSs are indicated by red arrows.