Fig. 6

Influence of the Pr77 secondary structure on the binding capacity of rTBP to double-stranded and single-stranded Pr77 sequences. EMSA measuring the binding capacity of rTBP to a native and DMSO-denatured ³²P-dsPr77 and to b native and heat-denatured sense- and antisense-single strands of the Pr77 sequence. a rTBP (6.35 μM) was incubated with 0.33 nM ³²P-dsPr77 in the presence of DMSO at 1, 3, 5 and 10% (v/v) at 37 °C for 30 min. Protein-DNA binding reactions without DMSO (−) and reactions carried out with ³²P-dsPr77 incubated with the same amounts of DMSO were included as controls. b rTBP (6.35 μM) was incubated with ³²P-labelled native or heat-denatured (95 °C for 5 min) sense (s) and antisense (as) DNA strands corresponding to the Pr77 sequence for 15 min at 4 °C. The reaction carried out with ³²P-dsPr77 (ds) was also included. All reactions in a and b were also performed in the absence of rTBP. Reactions were resolved in 6% polyacrylamide native gels. The black arrowhead indicates the formed complexes of reduced mobility, and the asterisks indicate the electrophoretic mobility of the nucleic acid-free conformations (dsPr77 and ssPr77)