Figure 1.

Site-specific DNA cleavage within open complexes formed by the holoenzyme carrying FeBABE-tethered S309C mutant α subunit. DNA fragments (213 bp) containing either T7D or rrnBP1 promoter (from –131 to +82) were ³²P-labeled at position –131 of the top strand. Open complexes were formed with the RNA polymerase containing of FeBABE conjugated S309C mutant α subunit. The DNA cleavage reaction was performed as described in Materials and Methods. Lane (–) represents DNA (6 nM) treated with reducing agents in the absence of RNA polymerase while lane (+) shows DNA extracted from the open complex (formed in the presence of 20 µM modified RNA polymerase) treated with reducing agents. Lane M represents a T-sequencing ladder of the primer extension reaction. Phosphodiester bonds specifically cleaved by S309C-conjugated FeBABE are indicated by arrows on the right. The numbers on the left indicate some positions of thymines. Open circles on right indicate bonds, which became hyperreactive to radicals upon open complex formation. A band generated by spontaneous nicking is marked by caret on the left. Primers (when used) are indicated by asterisk on the left.