Interaction of the motor SpoIIIE along a DNA with the specific sequence (SRS) in the presence of ATP. A single molecule dynamic predicted by a mathematical model is shown. The model assumes that the motor can bind/unbind, diffuse and translocate along the DNA. The blue shadowed region indicates the position of the specific sequence. In red and green the translocation and binding/diffusion events are shown. White represent unoccupied DNA sites.
Bacteria can exchange DNA as a mechanism of adaptation and survival. They use molecular motors capable of transporting DNA through cell membranes at high speed while maintaining a well-defined direction (that is, in a way, the bacteria having the DNA makes sure that the receiving bacteria get a complete copy of the DNA once the process was initiated). The bacterium Bacillus subtilis has the amazing capacity of creating a new version of itself, capable of survival during tens of years in a “dormant” state, provided that the environmental conditions are hostile. To do that, B. subtilis performs a process known as sporulation where a complete DNA copy is rapidly transported towards a daughter cell. The daughter cell (known as forespore) has a size significantly lower than the mother cell and its content and membrane will be adapted to confer resistance to numerous external perturbations (such as temperature changes, dehydration or radiations). To ensure that this process is carried on successfully, the bacteria uses a molecular motor, the protein SpoIIIE, capable of transporting DNA at high speeds by using the energy provided by ATP hydrolysis.
The mechanism by which this motor allows to bind the chromosome and translocate with the required direction and velocity has not yet been completely elucidated. In a recent study published in Scientific Reports , we (Osvaldo Chara and Augusto Borges from SysBio), in collaboration with Diego I. Cattoni, Pierre‐Emmanuel Milhiet and Marcelo Nöllmann , from the Centre de Biochimie Structurale (CNRS/INSERM/UM), Montpellier, France, made a key step in the understanding of the mechanism by which this molecular motor works.
In a previous article in EMBO Reports, the collaboration between SysBio and the CBS at Montpellier allowed us to determine that, in the absence of ATP, the molecular motor SpoIIIE is capable of rapidly exploring the DNA by linear diffusion to reach specific sequences that inform the direction of DNA transport (that is, sort of signals working as traffic lights). Once SpoIIIE is bound to these sequences, the mechanism that stabilizes this interaction is regulated by the balance between the dissociation rates at the specific and non-specific DNA sequences.
In our new study published in Scientific Reports, we investigated whether the specific DNA sequences regulate the direction of DNA by recruiting and orienting SpoIIIE or if they simply catalyze their translocation activity. We used atomic force microscopy and single-round fast kinetics translocation assays to determine the localization, diffusing and translocating dynamics of SpoIIIE complexes on DNA with or without specific sequences. We then combined these results with mathematical modeling to uncover that SpoIIIE controls the direction of DNA transport direction through the catalytic regulation of its motor activity by specific sequences.
In that way, SpoIIIE recognizes the specific sequences determining the translocation direction and its activation probability is enhanced several orders of magnitude. Moreover, we found that SpoIIIE can initiate translocation from non-specific DNA sequences, thanks to an alternative active search mechanism for the specific sequences located beyond the diffusion-driven exploratory distances. The mechanisms by which these molecular motors work have great interest for the biological community working in different areas, since they belong to a great molecular family that includes translocases and helicases. These proteins have important roles, not only in the DNA transport but also in replication, recombination and reparation of DNA, genic expression regulation, maturation and transport of RNA and many others.
This collaborative project is being funded by the ECOS grant between the groups leaded by Osvaldo Chara and Marcelo Nöllmann.
Interaction of the motor SpoIIIE along a DNA with the specific sequence (SRS) in the absence of ATP predicted by the previously described mathematical model are shown. The upper and middle panel show the binding and sliding of the motor both in real time (A) and the cumulative (B) respectively. Five representative DNAs are used. Bottom panel shows the frequency of motors binding to the specific sequence as time passes.