FIG. 5.
Interaction of the transcriptional components with the tRNAVal gene. The DNA fragment was incubated with the components (25 pmol of aTBP, 8 pmol of TFB, 0.8 pmol of RNA polymerase) as indicated at the bottom of each chromatogram for 20 min at 37°C, followed by DNase I treatment. Further treatment was as described in Materials and Methods. For size calibration of the peaks of the template strand, size markers with a different fluorescent dye were added to each probe and size calling was done by using the global Southern method according to the instructions of the supplier. The calculation of the fragment length was calibrated using sequencing reactions generated with the fluorescence-labeled primer. For the analysis of the nontemplate strand, the 220-bp DNA fragment was also generated by PCR using a biotinylated M13 reverse primer and a fluorescent-dye (ABI JOE)-labeled M13 primer. The DNA sequences of the plasmid pIC-31/30PRO-C25 are shown on the tops of the chromatograms in such a way that the peaks of the chromatograms can be directly correlated to the individual base positions within the DNA sequence. The promoter and the transcription start site are underlined. *, DNase I hypersensitive sites.