Figure 5. KMnO4 probing of roadblocked complex reveals upstream translocation of transcription bubble.
(a) and (b) Open complex was formed on P2∇5 DNA in the presence of GalR and RNA poymerase in the presence (lanes 2, 8) and absence (lanes 1, 7) of NTPs. Reactions were treated with KMnO4. DNA was purified and amplified using primer complementary to either the nontemplate (a) or template (b) strand. The same primers were used for sequencing the nontemplate (lanes 3–6) and template (9–12) strands. Note that in the lanes marked C, T, A and G reflect sequencing reactions using dideoxy G, A, T and C, respectively. Arrows on the left of the gels indicate the sites sensitive to KMnO4. (c) The sequence of the P2∇5 DNA shows KMnO4-sensitive residues in bold.