Nucleoid-associated proteins (NAPs) In eukaryotes, genomic DNA is condensed in the form of a repeating array of DNA-protein particles called
nucleosomes. A nucleosome consists of ~146 bp of DNA wrapped around an octameric complex of the
histone proteins. Although bacteria do not have histones, they possess a group of DNA binding proteins referred to as nucleoid-associated proteins (NAPs) that are functionally analogous to histones in a broad sense. NAPs are highly abundant and constitute a significant proportion of the protein component of nucleoid. A distinctive characteristic of NAPs is their ability to bind DNA in both a specific (either sequence- or structure-specific) and non-sequence specific manner. As a result, NAPs are dual function proteins. the most extensively studied of which are HU, IHF, H-NS, and Fis. Their abundance and DNA binding properties and effect on DNA condensation and organization are summarized in the tables below. HU exists in
E. coli as homo- and heterodimers of two subunits HUα and HUβ sharing 69% amino acid identity. Although it is referred to as a histone-like protein, close functional relatives of HU in eukaryotes are
high-mobility group (HMG) proteins, and not histones. HU is a non-sequence specific DNA binding protein. It binds with low-affinity to any linear DNA. However, it preferentially binds with high-affinity to a structurally distorted DNA. Examples of distorted DNA substrates include
cruciform DNA, bulged DNA, dsDNA containing a single-stranded break such as
nicks, gaps, or
forks. Furthermore, HU specifically binds and stabilizes a protein-mediated DNA loop. In the structurally specific DNA binding mode, HU recognizes a common
structural motif defined by bends or kinks created by distortion, whereas it binds to a linear DNA by locking the phosphate backbone. While the high-affinity structurally-specific binding is required for specialized functions of HU such as
site-specific recombination,
DNA repair,
DNA replication initiation, and gene regulation, it appears that the low-affinity general binding is involved in DNA condensation. The following
in vitro studies suggest possible mechanisms of how HU might condense and organize DNA
in vivo. Not only HU stably binds to distorted DNA with bends, it induces flexible bends even in a linear DNA at less than 100 nM concentration. In contrast, HU shows the opposite architectural effect on DNA at higher physiologically relevant concentrations. It forms rigid
nucleoprotein filaments causing the straitening of DNA and not the bending. The filaments can further form a DNA network (DNA bunching) expandable both laterally and medially because of the HU-HU multimerization triggered by the non-sequence-specific DNA binding. This indicates that the flexible bends are more likely to occur
in vivo. The flexible bending would cause condensation due to a reduction in the
persistence length of DNA as shown by
magnetic tweezers experiments, which allow studying condensation of a single DNA molecule by a DNA binding protein. However, because of the
cooperativity, the rigid filaments and networks could form in some regions in the chromosome. The filament formation alone does not induce condensation, but behaves differently from HU in many aspects. Unlike HU, which preferentially binds to a structural motif regardless of the sequence, IHF preferentially binds to a specific DNA sequence even though the specificity arises through the sequence-dependent DNA structure and deformability. The specific binding of IHF at cognate sites bends DNA sharply by >160-degree. The estimated abundance of IHF in the growth phase is about 6000 dimers per cell. Assuming that one IHF dimer binds to a single motif and nucleoid contains more than one genome equivalent during the exponential growth phase, most of the IHF molecules would occupy specific sites in the genome and likely only condense DNA by inducing sharp bending. Unlike HU, IHF does not form thick rigid filaments at higher concentrations. Instead, its non-specific binding also induces DNA bending albeit the degree of bending is much smaller than that at specific sites and is similar to the flexible bending induced by HU in a linear DNA at low concentrations.
In vitro, the bending induced by non-specific binding of IHF can cause DNA condensation and promotes the formation of higher-order nucleoprotein complexes depending on the concentrations of potassium chloride and
magnesium chloride. from other NAPs is the ability to switch from the homodimeric form at relatively low concentrations (−5 M) to an oligomeric state at higher levels. Because of oligomerization properties, H-NS spreads laterally along AT-rich DNA in a
nucleation reaction, where high-affinity sites function as nucleation centers. The spreading of H-NS on DNA results in two opposite outcomes depending on the magnesium concentration in the reaction. At low magnesium concentration ( 5 mM). The formation of rigid filaments results in straightening of DNA with no condensation whereas the bridging causes substantial DNA folding. Although H-NS has been demonstrated to prefer curved DNA formed by repeated A-tracks in DNA sequences the basis of the selective binding is the presence of a
conserved sequence motif found in AT-rich regions. More importantly, the frequent occurrence of the sequence motif within an H-NS binding region that can re-enforce the cooperative protein-protein interactions, and the unusually long length of the binding region are consistent with the spreading of the protein. Whether the filament formation or DNA bridging is prevalent
in vivo depends on the physiological concentration of magnesium inside the cell. If the magnesium concentration is uniformly low (1 mM. The loops in magnetic tweezers experiments are distinct from the micro-loops created by coherent DNA bending at cognate sites, as they require the formation of high-density DNA-protein complexes achieved by sequence-independent binding. Although, occurrence of such loops
in vivo remains to be demonstrated, high-density binding of Fis may occur
in vivo through concerted action of both specific and non-specific binding. The in-tandem occurrence of specific sites might initiate a nucleation reaction similar to that of H-NS, and then non-specific binding would lead to the formation of localized high-density Fis arrays. The bridging between these localized regions can create large DNA loops. Thus, any role in chromosomal condensation by Fis must be specific to growing cells. Moreover, treatment with RNase A disrupted the DNA fibers into thinner fibers, as observed by an
atomic force microscopy of the nucleoid using the "on-substrate lysis procedure". These findings demonstrated the participation of RNA in the nucleoid structure, but the identity of the RNA molecule(s) remained unknown until recently. Moreover, HU preferentially binds to RNA containing secondary structures and an RNA-DNA hybrid in which the RNA contains a nick or overhang. The binding affinities of HU with these RNA substrates are similar to those with which it binds to distorted DNA. An
immunoprecipitation of HU-bound RNA coupled to reverse transcription and microarray (RIP-Chip) study as well as an analysis of RNA from purified intact nucleoids identified nucleoid-associated RNA molecules that interact with HU. Recent studies provide insights into the molecular mechanism of how naRNA4 establishes DNA-DNA connections. The RNA targets regions of DNA containing cruciform structures and forms an RNA-DNA complex that is critical for establishing DNA-DNA connections. Surprisingly, although HU helps in the formation of the complex, it is not present in the final complex, indicating its potential role as a catalyst (chaperone). The nature of the RNA-DNA complex remains puzzling because the formation of the complex does not involve extensive Watson/Crick base pairing but is sensitive to RNase H, which cleaves RNA in an RNA-DNA hybrid and the complex binds to an antibody specific to RNA-DNA hybrids. The number of times the two strands cross each other in a topologically constrained DNA is called the
linking number (Lk), which is equivalent to the number of helical turns or twists in a circular molecule. The Lk of a
topological DNA remains invariant, no matter how the DNA molecule is deformed, as long as neither strand is broken. The Lk of DNA in the relaxed form is defined as Lk0. For any DNA, Lk0 can be calculated by dividing the length (in bp) of the DNA by the number of bp per helical turn. This is equal to 10.4 bp for the relaxed
B-form DNA. Any deviation from Lk0 causes
supercoiling in DNA. A decrease in the linking number (Lk0) creates negative supercoiling whereas an increase in the linking number (Lk>Lk0) creates positive supercoiling. The supercoiled state (when Lk is not equal to Lk0) results in a transition in DNA structure that can manifest as a change in the number of twists (negative 10.4 bp per turn) and/or in the formation of
writhes, called supercoils. Thus, Lk is mathematically defined as a sign dependent sum of the two geometric parameters, twist and writhe. A quantitative measure of supercoiling that is independent of the size of DNA molecules is the supercoiling density (σ) where σ =∆Lk/Lk0. In the eukaryotic
chromatin, DNA is found mainly in the toroidal form that is restrained and defined by histones through the formation of nucleosomes. In contrast, in the
E. coli nucleoid, about half of the chromosomal DNA is organized in the form of free, plectonemic supercoils. The remaining DNA is restrained in either the plectonemic form or alternative forms, including but not limited to the toroidal form, by interaction with proteins such as NAPs. Thus, plectonemic supercoils represent effective supercoiling of the
E. coli genome that is responsible for its condensation and organization. Both plectonemic and toroidal supercoiling aid in DNA condensation. Branching of plectonemic structures provides less DNA condensation than does the toroidal structure. A same size DNA molecule with equal supercoiling densities is more compact in a toroidal form than in a plectonemic form. In addition to condensing DNA, supercoiling aids in DNA organization. It promotes disentanglement of DNA by reducing the probability of catenation. Supercoiling also helps bring two distant sites of DNA in proximity thereby promoting a potential functional interaction between different segments of DNA.
E. coli possesses four topoisomerases.
DNA gyrase introduces negative supercoiling in the presence of ATP and it removes positive supercoiling in the absence of ATP. Across all forms of life, DNA gyrase is the only topoisomerase that can create negative supercoiling and it is because of this unique ability that bacterial genomes possess free negative supercoils; DNA gyrase is found in all bacteria but absent from higher eukaryotes. In contrast, Topo I opposes DNA gyrase by relaxing the negatively supercoiled DNA. There is genetic evidence to suggest that a balance between the opposing activities of DNA gyrase and Topo I are responsible for maintaining a steady-state level of average negative superhelicity in
E. coli. Both enzymes are essential for
E. coli survival. A null strain of
topA, the gene encoding Topo I, survives only because of the presence of suppressor mutations in the genes encoding DNA gyrase. The primary function of Topo IV is to resolve sister chromosomes. However, it has been shown to also contribute to the steady-state level of negative supercoiling by relaxing negative supercoiling together with Topo I.
Transcription A twin supercoiling domain model proposed by Liu and Wang argued that unwinding of
DNA double helix during transcription induces supercoiling in DNA as shown in. According to their model, transcribing
RNA polymerase (RNAP) sliding along DNA forces the DNA to rotate on its helical axis. A hindrance in the free rotation of DNA might arise due to a topological constraint, causing the DNA in front of RNAP to become over-twisted (positively supercoiled) and the DNA behind RNAP would become under-twisted (negatively supercoiled). It has been found that a topological constraint is not needed because RNAP generates sufficient torque that causes supercoiling even in a linear DNA template. If DNA is already negatively supercoiled, this action relaxes existing negative supercoils before causing a buildup of positive supercoils ahead of RNAP and introduces more negative supercoils behind RNAP. In principle, DNA gyrase and Topo I should remove excess positive and negative supercoils respectively but if the RNAP elongation rate exceeds the turnover of the two enzymes, transcription contributes to the steady-state level of supercoiling. They can do so either by changing the helical pitch of DNA or generating toroidal writhes by DNA bending and wrapping. Alternatively, NAPs can preferentially bind to and stabilize other forms of the underwound DNA such as cruciform structures and branched plectonemes. Fis has been reported to organize branched plectonemes through its binding to cross-over regions and HU preferentially binds to cruciform structures. There is genetic evidence to suggest that HU controls supercoiling levels by stimulating DNA gyrase and reducing the activity of Topo I. In support of the genetic studies, HU was shown to stimulate DNA gyrase-catalyzed decatenation of DNA
in vitro. It is unclear mechanistically how HU modulates the activities of the gyrase and Topo I. HU might physically interact with DNA gyrase and Topo I or DNA organization activities of HU such as DNA bending may facilitate or inhibit the action of DNA gyrase and Topo I respectively. In other words, a single cut in one domain will only relax that domain and not the others. A topological domain forms because of a supercoiling-diffusion barrier. Independent studies employing different methods have reported that the topological domains are variable in size ranging from 10 to 400 kb. A random placement of barriers commonly observed in these studies seems to explain the wide variability in the size of domains. NAPs such as H-NS and Fis are potential candidates, based on their DNA looping abilities and the distribution of their binding sites. (ii) Bacterial interspersed mosaic elements (BIMEs) also appear as potential candidates for domain barriers. BIMEs are palindromic repeats sequences that are usually found between genes. A BIME has been shown to impede diffusion of supercoiling in a synthetically designed topological cassette inserted in the
E. coli chromosome. There are ~600 BIMEs distributed across the genome, possibly dividing the chromosome into 600 topological domains. (iii) Barriers could also result from the attachment of DNA to the cell membrane through a protein which binds to both DNA and membrane or through nascent transcription and the translation of membrane-anchored proteins. (iv) Transcription activity can generate supercoiling-diffusion barriers. An actively transcribing RNAP has been shown to block dissipation of plectonemic supercoils, thereby forming a supercoiling-diffusion barrier. == Growth-phase dependent nucleoid dynamics ==