Probing the bridging behaviour of the Rok protein from Bacillus subtilis with quadruple-trap optical tweezers.

Abstract

Bacteria densely organize their genome in the form of a nucleoid stored in the cytoplasm. This compact, functional form of DNA is achieved by the use of nucleoid-associated proteins (NAPs), proteins that play a role in DNA organization as well as regulating gene expression. The various NAPs found across bacterial species are classed based on shared functions with well-studied NAPs (H-NS, HU, IHF and Fis). In Bacillus subtilis, the NAP Rok shares functional similarities with H-NS and is proposed to be a H-NS-like protein, however, some fundamental differences in binding activity and response to physio-chemical changes have been observed. The aim of this study is to investigate the structural, kinetic and dynamic attributes of Rok-DNA binding in order to better understand the role of Rok as a NAP in Bacillus subtilis. Dual-trap optical tweezer experiments, in which Rok was allowed to bind to one double stranded lambda DNA molecule, showed that Rok compacts DNA while no impact on the mechanical properties of DNA was observed. Using quadruple-trap optical tweezer experiments, Rok was allowed to bind to two double stranded lambda DNA molecules. In this tweezer configuration, shearing and unzipping experiments were used to probe the interaction of Rok with DNA and revealed formation of DNA-Rok-DNA bridges. We found that the Rok-mediated DNA compaction and bridging is likely not due to protein-protein interaction suggesting that one Rok protein is able to bind two DNA molecules or two sites on a single DNA molecule. Additionally, DNA-Rok bonds withstood considerably high forces before rupturing. Our results combined with recent literature suggest that Rok might not have a similar function to H-NS in Bacillus subtilis but that it rather acts as a stabilizer facilitating the function of other proteins.