Figure 1.

Functional characterization of the p21 and Fas/APO1 core promoters. (A) Schematic diagram of p21 and Fas/APO1 promoters, including their respective p53-binding sites. (B) In vitro transcription of p21 or Fas/APO1 plasmids was performed and analyzed by primer extension. (C) Schematic diagram of single- and multiple-round transcription in the presence of sarkosyl. Addition of sarkosyl to form PICs prevents new PIC formation. Templates without sarkosyl undergo multiple rounds of transcription. (D) Rates of PIC formation were analyzed by adding sarkosyl just before (time 0), or various times after, combining DNA templates with HeLa nuclear extracts (HNEs). Transcription was initiated by adding NTPs. (E) Quantification and graphical representation of D. (F) Rounds of transcription were analyzed by allowing PICs to form for 1 h before initiating transcription (time 0). The initiated reactions then underwent multiple rounds of transcription before sarkosyl addition at intervals from 15 sec to 30 min. The first lane shows that sarkosyl addition to DNA templates before exposure to HeLa extract prevents formation of functional PICs. (G) Rounds of transcription were also measured using DNA templates immobilized on magnetic beads. Extract and immobilized DNA were mixed for 1 h to allow PIC formation similar to C. For single-round transcription, the DNA template was washed with buffer followed by addition of NTPs to allow elongation, whereas this wash step was not included for multiple rounds of transcription. (H) Rounds of transcription were quantified, and the signal at each time point was compared with time 0 to calculate the rounds of reinitiation.