Figure 5

SRS enhances ATP-dependent translocation activity of SpoIIIE. (A) Scheme representing the triplex displacement experimental design. (B) Time course of triplex displacement by SpoIIIE for substrates with (green circles) or without (orange squares) SRS sequences in the presence of ATP. Inset shows that in the absence of ATP, SpoIIIE is unable to displace the triplex. (C) Effect of SRS in triplex displacement rates quantified as the ratio between the area under the kinetic traces for DNA _SRS and DNA _NS (θ _SRS/NS ). Blue squares and error bars indicate mean and standard deviation from the simulation results by varying p _ATP ^SRS while maintaining p _ATP ^NS constant at 0.012. Red circle depicts the result expected for the ‘recruitment and orient’ directionality model (see main text). Green dotted line and grey shaded area indicate the mean and standard deviation of θ _SRS/NS obtained from the experimental results shown in panel B (n = 3). Solid blue lines connecting squares are only a guide to the eye. Insets depict the time courses for triplex displacement obtained from simulations when p _ATP ^SRS = p _ATP ^NS (left) and p _ATP ^SRS = 0.8 (right). Parameter values for simulations are depicted in Table 1. (D) SpoIIIE in vivo directionality mechanism. Free, bound, diffusing and active SpoIIIE are represented in grey, black, dotted grey and filled light blue respectively. Dotted green and orange contour lines represent the two alternative pathways for SpoIIIE directionality regulation. Upon binding (black arrow) SpoIIE can explore DNA by diffusion (dotted grey arrow) and/or dissociate (red arrows) and/or become active (green arrows). Once active SpoIIIE can translocate (dashed black arrows). Thickness of red and green arrows is proportional to the rate/probability values obtained from experimental and modelling optimized parameter values when SpoIIIE is interacting with non-specific or SRS sequences. Upper scheme depicts a B. subtilis cell in the initial stages of sporulation. oriC regions (red circles) move towards the cell poles and after asymmetric division SpoIIIE (blue circles) is recruited to the septum to transport two-thirds of the chromosome into the forespore.