FIGURE 4.

DNase I footprinting provides evidence for assembly of initiation complexes on LSP and HSP1 oligo templates. DNase I footprinting was performed using LSP and HSP1 DNA oligonucleotide templates. Proteins were assembled on the indicated ³²P-labeled DNA oligo (2.5 μm) by combining one or more of the following components: h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm), as indicated, in reaction buffer at 32 °C. DNA cleavage was initiated by addition of RQ1 DNase (0.002 unit/μl) and CaCl₂ (1 mm) and quenched after 2 min by addition of stop/trap buffer. Products were resolved by denaturing PAGE on 8% gels and visualized by phosphorimaging. The locations/orientations of the h-mtTFA-binding site and transcription start site are indicated for the LSP and HSP1 promoters. A 10-bp ladder was used as a size marker (M). a, footprinting was performed on LSP and HSP1 containing ³²P in the 5′ end of the templating strand. b, quantification of selected lanes of the gels shown in a. The intensities were normalized to the −40 position of the control (lane 2 of LSP or HSP1 in a). Initiation complex refers to lane 6 of LSP or HSP1 in a. Asterisk refers to the location of the ³²P label.