Figure 2. Nanomolar RapA disrupts PTCs in an ATP-dependent manner.

(A) Schematic of the single-molecule rPTC disruption experiment. Circular npDNA^Cy5 molecules (tethered to a glass surface (blue)) contain no known promoter sequences and were photobleached (black star) after recording their locations. At time zero, RapA or RapA⁶⁵⁰ (orange) was added either together with RNAP⁵⁴⁹ (green; pre-mix) or after free RNAP⁵⁴⁹ was removed from the chamber (washout). Presence at the DNA location of RNAP⁵⁴⁹ is detected by the fluorescence from the green-excited dye molecule (star) under continuous laser excitation. Time resolution 1 s; 532 nm laser power 400 µW; RNAP⁵⁴⁹ photobleaching rate (Fig. S3) 0.0006 s⁻¹. (B) Example RNAP⁵⁴⁹ fluorescence intensity record at the location of a single DNA molecule from a washout experiment, plotted as in Fig. 1B–D. (C) Reciprocal of the mean RNAP⁵⁴⁹ dwell time τ at various RapA or RapA⁶⁵⁰ concentrations. Experiments were conducted using the pre-mix or washout protocols in the presence or absence of 1 mM ATP. Data were fit to hyperbolic (solid line) or linear (dashed lines) models yielding fit parameters given in Table S1. Inset: Magnified view of the low concentration range with additional data at zero RapA.