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Regulation of protein mobility is a fundamental aspect of cellular processes. In this study, we examined the impact of DNA methylation on the diffusion of nucleoid associated protein Hfq. This protein is one of the most abundant proteins that shapes the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy was employed to monitor the movement of Hfq along double-stranded DNA, which was stretched due to confinement within a nanofluidic channel. The mobility of Hfq is significantly influenced by DNA methylation. Our results underscore the importance of bacterial epigenetic modifications in governing the movement of nucleoid associated proteins such as Hfq. Increased levels of methylation result in enhanced binding affinity, which in turn slows down the diffusion of Hfq on DNA. The reported control of protein mobility by DNA methylation has potential implications for the mechanisms involved in target DNA search processes and dynamic modelling of the bacterial chromosome.

The mobility of protein is fundamental in the machinery of life. Here, we have investigated the effect of DNA binding in conjunction with DNA segmental fluctuation (internal motion) of the bacterial Hfq master regulator devoid of its amyloid C-terminus domain. Hfq is one of the most abundant nucleoid associated proteins that shape the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy has been used to track a C-terminus domain lacking mutant form of Hfq on double-stranded DNA, which is stretched by confinement to a rectangular nanofluidic channel. The mobility of the mutant is strongly accelerated with respect to the wild-type variant. Furthermore, it shows a reverse dependence on the internal motion of DNA, in that slower motion results in slower protein diffusion. The results demonstrate the subtle role of DNA internal motion in controlling the mobility of a nucleoid associated protein, and, in particular, the importance of transient binding and moving DNA strands out of the way.

Protein transport on DNA is at the core of the machinery of life. Here we investigated the influence of DNA internal motion on the mobility of Hfq, which is involved in several aspects of nucleic acid metabolism and is one of the nucleoid-associated proteins that shape the bacterial chromosome. Fluorescence microscopy was used to follow Hfq on double-stranded DNA that was stretched by confinement to a channel with a diameter of 125 nm. The protein mobility shows a strong dependence on the internal motion of DNA in that slower motion results in faster protein diffusion. A model of released diffusion is proposed that is based on three-dimensional diffusion through the interior of the DNA coil interspersed by periods in which the protein is immobilized in a bound state. We surmise that the coupling between DNA internal motion and protein mobility has important implications for DNA metabolism and protein-binding-related regulation of gene expression.

Molecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Escherichia coli Hfq is reported, involved in controlling the flow of a DNA solution. A combination of experimental methodologies has been used to probe the interaction of Hfq with DNA and to measure the rheological properties of the complex. A physical gel with a temperature reversible elasticity modulus is formed due to the formation of noncovalent cross-links. The mechanical response of the complexes shows that they are inhomogeneous soft solids. Our experiments indicate that the Hfq C-terminal region could contribute to the genome's mechanical response. The reported viscoelasticity of the DNA-protein complex might have implications for cellular processes involving molecular transport of DNA or segments thereof.