Fig. 2

Identification of TcpK binding site. a Gel mobility shift assays with His₆-TcpK. The DIG-labeled 150 bp oriT fragment was incubated alone, in the presence of 450 nM TcpM, in the presence of 750 nM TcpK or in the presence of 450 nM TcpM and TcpK (50 nM), as indicated. Reactions were separated on a 6% (w v⁻¹) TBE acrylamide gel for 60 min at 200 V at 4 °C. Open arrows indicate free DNA (F), protein–DNA complexes (C), protein–DNA super-shifted complexes (S) and complexes in the wells (W). b SPR analysis of TcpK binding to oriT. The 150 bp oriT site of pCW3 was divided into overlapping fragments (oriT#1–oriT#13) each comprised of 30 bp with 20 bases overlap from one fragment to the next. The biotinylated single-stranded DNA (ssDNA) ReDCaT linker was immobilized on the surface of a Streptavidin (SA) chip to a surface density of 450 Resonance Unit (RU). c The binding of TcpK to the oligonucleotides was tested by SPR at protein concentrations of 10 μM (checked bars) and 1 μM (solid bars). The binding levels are expressed as a percentage of the theoretical maximum response (see Methods). d SPR-based mapping defines the TcpK binding site. A 39 bp fragment that encompassed oriT#1 and oriT#2 was used as the initial test fragment (L2R1 and R2L1 for left to right and right to left, respectively). Subsequent oligonucleotides (L2R2 to L2R12 and R2L2 to R2L12) were incrementally shorter by 2 nt. TcpK binding to each oligonucleotide L2R (e) and R2L (f) was tested at protein concentrations of 1 μM (checked bars) and 0.1 μM (solid bars). The binding levels are expressed as a percentage of the theoretical maximum response