Abstract:HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influ… Show more
“…This is in agreement with earlier observations that increasing ionic strength reduces HU binding affinity 30 . Overall our results agree with earlier observations 22 , 26 , 33 and indicate switching between two modes of binding depending on HU concentration. Figure 3 Contour length (Lc) and persistence length (Lp) as a function of protein concentration.…”
Section: Resultssupporting
confidence: 93%
“…Clusters of fluorescent signals of proteins appear along the timeline in the condition without Mg 2+ and at tension above 10 pN, while no evidence for clusters is found with 8 mM Mg 2+ . In our earlier studies 33 we showed that HU dimers bind DNA with high cooperativity (both in the absence and presence of 8 mM Mg 2+ ) when there is no tension on the DNA. Similar binding behaviour of HU is expected at tension under 10 pN since the extension of the DNA is shorter than Lc below that force and thus somewhat comparable to the condition in the TPM experiment.…”
Section: Resultsmentioning
confidence: 86%
“…The diffusion coefficients measured in these studies are not very similar: the diffusion coefficient is five times higher in vitro than in vivo (0.5 μm s −1 in vitro vs 0.1 μm s −1 in vivo). This discrepancy is attributed to different ionic conditions and the complexity of the in vivo medium with the numerous macromolecules present in the cytoplasm resulting in crowding, which likely influences the DNA binding properties of HU 33 . However, both studies underline the dynamic nature of the bound HU on DNA.…”
Architectural DNA–binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA–binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg2+; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg2+ in solution. Additionally, we investigated the effect of DNA tension on HU–DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg2+ increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg2+ compared to without Mg2+ suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.
“…This is in agreement with earlier observations that increasing ionic strength reduces HU binding affinity 30 . Overall our results agree with earlier observations 22 , 26 , 33 and indicate switching between two modes of binding depending on HU concentration. Figure 3 Contour length (Lc) and persistence length (Lp) as a function of protein concentration.…”
Section: Resultssupporting
confidence: 93%
“…Clusters of fluorescent signals of proteins appear along the timeline in the condition without Mg 2+ and at tension above 10 pN, while no evidence for clusters is found with 8 mM Mg 2+ . In our earlier studies 33 we showed that HU dimers bind DNA with high cooperativity (both in the absence and presence of 8 mM Mg 2+ ) when there is no tension on the DNA. Similar binding behaviour of HU is expected at tension under 10 pN since the extension of the DNA is shorter than Lc below that force and thus somewhat comparable to the condition in the TPM experiment.…”
Section: Resultsmentioning
confidence: 86%
“…The diffusion coefficients measured in these studies are not very similar: the diffusion coefficient is five times higher in vitro than in vivo (0.5 μm s −1 in vitro vs 0.1 μm s −1 in vivo). This discrepancy is attributed to different ionic conditions and the complexity of the in vivo medium with the numerous macromolecules present in the cytoplasm resulting in crowding, which likely influences the DNA binding properties of HU 33 . However, both studies underline the dynamic nature of the bound HU on DNA.…”
Architectural DNA–binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA–binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg2+; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg2+ in solution. Additionally, we investigated the effect of DNA tension on HU–DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg2+ increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg2+ compared to without Mg2+ suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.
“…The simplest models adopt single proteins, usually chosen among those known to have a low tendency to interact. Examples of these proteins are the bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, and bovine serum albumin (BSA). − Studies of mixed crowders were also conducted, and the advantages of mixed crowding over homogeneous crowding were independently suggested by different groups. − Zhou tested, for instance, the effects of mixed crowding on protein stability and suggested that optimal crowding effects could be obtained by adjusting mixing ratios between crowders’ populations . Shah et al also suggested a role for enthalpic interactions in mixtures using an in silico approach at lower than physiological temperatures (27 °C) .…”
It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.
“…It may be useful here to distinguish between the terms 'macromolecular crowding' and 'macromolecular con nement': the dynamic effects of volume exclusion are caused in the former by other macromolecules and in the latter by the static shape and size of the system (Lecinski et al, 2021). It may also be useful to distinguish between different crowding agents, as shown for the compaction and DNA-binding of HU (Lin et al, 2020) and, in particular, to distinguish between molecular crowding and macromolecular crowding, which can affect water dynamics differently (Verma et al, 2018). Indeed, in highly crowded media, molecular crowding affects both the interstices between the crowder molecules and the interfacial water, whilst macromolecular crowding does not affect the bulk water and only has a small effect on the interfacial water (Verma et al, 2018).…”
Section: Molecular and Macromolecular Crowding Or Water Availabilitymentioning
A fundamental problem in biology is how cells obtain the reproducible, coherent phenotypes needed for natural selection to act or, put differently, how cells manage to limit their exploration of the vastness of phenotype space. A subset of this problem is how they regulate their cell cycle. Bacteria, like eukaryotic cells, are highly structured and contain scores of hyperstructures or assemblies of molecules and macromolecules. The existence and functioning of certain of these hyperstructures depend on phase transitions. Here, I propose a conceptual framework to facilitate the development of water-clock hypotheses in which cells use water to generate phenotypes by living ‘on the edge of phase transitions’. I give an example of such a hypothesis in the case of the bacterial cell cycle and show how it offers a relatively novel ‘view from here’ that brings together a range of different findings about hyperstructures, phase transitions and water and that can be integrated with other hypotheses about differentiation, metabolism and the origins of life.
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