Abstract
Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.
Highlights
Bacterial DNA pumps are responsible for the intra and intercellular transfer of genetic material across membranes during sporulation, cell division and conjugation[1]
Different studies agree that SpoIIIE activity is stimulated by SRS11,12, the capacity of SpoIIIE to efficiently couple ATP hydrolysis to translocation in absence of specific octameric sequences (SRS) remains unclear
To directly test the ability of SpoIIIE to couple ATP hydrolysis to translocation independently of the presence of SRS, we performed atomic force microscopy (AFM) imaging of SpoIIIE incubated with linearized DNA substrates without or with SRS sequences oriented towards the nearest DNA end (DNANS and DNASRS respectively) in the presence and absence of ATP
Summary
Bacterial DNA pumps are responsible for the intra and intercellular transfer of genetic material across membranes during sporulation, cell division and conjugation[1]. It has been shown that SpoIIIE has higher affinity for SRS than for non-specific DNA and that SRS stimulates its ATPase activity[11,12], suggesting that SRS could either regulate directionality by recruiting and orienting SpoIIIE hexamers prior to engaging translocation or by triggering SpoIIIE translocation. The distinction between these two mechanisms (recruitment and orienting vs catalytic triggering) requires direct observation of SpoIIIE-DNA complexes and discrimination between the translocating state when complexes are interacting with non-specific DNA or with SRS. We study how sequence recognition regulates the directionality and translocation activity of SpoIIIE by combining single molecule imaging with single round translocation measurements and mathematical modelling. Our results strongly suggest that rather than recruiting and orienting the non-active protein, SRS sequences regulate directionality by modulating the translocation activity of SpoIIIE
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