PMC

Hexameric SpoIIIE binds to non-specific DNA and finds SRS by an ATP-independent mechanism. (A–C) AFM imaging of SpoIIIE–DNA complexes. Yellow arrow indicates free SpoIIIE, orange arrows indicate SpoIIIE bound to non-specific DNA sequences and green arrows indicate SpoIIIE–SRS complexes. (A) T_NS substrates incubated with SpoIIIE for 15 min. (B–C) T_SRS substrates incubated with SpoIIIE for 2 and 15 min, respectively. (D–F) Histograms of SpoIIIE distributions on DNA for the different substrates and incubation times described in A–C. SpoIIIE position in DNA was measured as the minimum distance of SpoIIIE particles to the end of DNA. A total of 35–40 individual SpoIIIE–DNA complexes were analysed for each condition. Dashed line indicates the average frequency of SpoIIIE complexes expected for an ideally homogenous distribution in T_NS substrates. Insets in D–F show the simulated SpoIIIE distributions in different DNA substrates obtained from Monte Carlo modelling using model II. Detailed description and simulations parameters are given in online and online.

Proposed mechanism for sequence-directed SpoIIIE directional translocation. (A) Relative ATPase activity as a function of DNA concentration. Solid line represents fit of equation S9 ( online) to data with V_max=2.6±0.1 for DNA_NS and 4.1±0.3 for DNA_SRS. Relative ATPase activities are obtained by normalizing the ATPase activity of SpoIIIE on DNA_SRS or DNA_NS by the ATPase activity in saturating amounts of λ-DNA (≈10 mol ATP per second per mol SpoIIIE). (B) Schematic representation of the target search and sequence-specific activation model (see main text). (B-iv) Schematic representation of a SpoIIIE hexamer binding SRS and structural elements proposed to be involved in sequence-specific activation of the motor (see online for high-resolution model). Stimulation helix is shown in green, subunit coordination loop in dark green and α jaw regulation loop in brown. (B-v) Actively translocating SpoIIIE can interact with non-permissive SRS sequences leading to SpoIIIE dissociation [] or translocation reversal [, ].

Pre-formed SpoIIIE hexamers bind specifically but do not load onto SRS. (A) Scheme representing the preferential loading model proposed for FtsK/SpoIIIE sequence recognition and translocation mechanism. (B) Scheme representing the experimental set-up for fluorescence anisotropy measurements of SpoIIIE–DNA-binding equilibrium and kinetics. (C) SpoIIIE-binding isotherms for DNA_SRS and DNA_NS. Solid line represents the fitting of equation S2 ( online) to experimental data (see best fitting parameters in main text). Inset shows affinity changes of SpoIIIE for DNA_SRS and DNA_NS versus ionic strength (I) of monovalent salt (NaCl). (D) On-rate kinetics of SpoIIIE binding to DNA_SRS and DNA_NS. (E) Off-rate kinetics of SpoIIIE dissociation from DNA_SRS and DNA_NS. Solid lines in D and E represent the fit of equation S3 and S4 ( online) to experimental data (see main text for best fitting parameters). (F) Diffusion coefficients of Cy5-labelled SpoIIIE versus protein concentration in presence (grey triangles) or absence (black squares) of DNA_SRS obtained from FCS measurements. Solid line represents the fitting of model M1 to the experimental data (see main text for fitting parameters). In panels C and F, error bars represent s.d. with n=5 and 3, respectively.