FIG. 4.

Open complex formation and transcription at low temperatures. Transcription reactions for open complex formation were performed as described in Materials and Methods. Linearized (A) or negatively supercoiled (B) plasmid pIC-31/30PRO-C25 was incubated with the components indicated at the bottom of each panel for 20 min at the temperatures indicated. After treatment with KMnO₄, the hyperreactive sites on the nontemplate strand were analyzed by asymmetric PCR using a fluorescent-dye-labeled primer. For fragment size calibration of the peaks, size markers with a different fluorescent dye were added to each probe and size correlation was done by using the global Southern method according to the instructions of the supplier. The DNA sequences of the plasmid pIC-31/30PRO-C25 are shown on top in such a way that the peaks of the chromatograms can be directly correlated to the individual base positions within the DNA sequence. The transcription start site is underlined. (C) Transcription reaction mixtures were incubated for 20 min at the temperatures indicated on top of the lanes using negatively supercoiled (C) or linearized (L) DNA.