Abstract
Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF–DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: ‘associated’ (73° of DNA bend), ‘half-wrapped’ (107°) and ‘fully-wrapped’ (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.
Highlights
Nucleoid-associated proteins (NAPs) are a collection of DNA-interacting proteins that perform crucial roles of organization, packaging and gene regulation in prokaryotic chromosomes [1, 2], including functions as transcription factors [3]
Multimodality of Integration host factor (IHF) specific and non-specific binding revealed by atomic force microscopy (AFM) To investigate the differences between specific and non-specific binding, two short sequences were amplified from phage λ, one with no IHF consensus sequence (0λ361) and another with a single consensus sequence (1λ306) of lengths 361 bp and 306 bp respectively (Figure 2)
DNA contours were recovered by skeletonizing pre-processed AFM images, with qualitatively different behavior observed depending upon the presence of IHF (Figures 3)
Summary
Nucleoid-associated proteins (NAPs) are a collection of DNA-interacting proteins that perform crucial roles of organization, packaging and gene regulation in prokaryotic chromosomes [1, 2], including functions as transcription factors [3]. They often bind non-depending on their relative concentration in cells, which vary across orders of magnitude depending on cell-cycle stages and environment conditions [4]. The removal of IHF caused a 50% reduction in biofilm thickness [22] and IHF has been imaged at vertices of an extracellular DNA lattice [21]
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