Background
In many bacteria, theCondensation and organization
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 theHU
Histone-like protein from ''E. coli'' strain U93 (HU) is an evolutionarily conserved protein in bacteria. 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 asIHF
Integration host factor (IHF) is structurally almost identical to HU 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. An occurrence of the cognate sequence motif is about 3000 in the ''E. coli'' genome. 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. Besides preferential binding to a specific DNA sequence, IHF also binds to DNA in a non-sequence specific manner with the affinities similar to HU. A role of the non-specific binding of IHF in DNA condensation appears to be critical in the stationary phase because the IHF abundance increases by five-fold in the stationary phase and the additional IHF dimers would likely bind the chromosomal DNA non-specifically. 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. The higher-order DNA organization by IHF ''in vivo'' is as yet unclear.H-NS
A distinguishable feature of histone-like or heat-stable nucleoid structuring protein (H-NS) from other NAPs is the ability to switch from the homodimeric form at relatively low concentrations (<1 x 10−5 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 (< 2 mM), H-NS forms rigid nucleoprotein filaments whereas it forms inter- and intra-molecular bridges at higher magnesium concentrations (> 5 mM). The formation of rigid filaments results in straightening of DNA with no condensation whereas the bridging causes substantial DNA folding. Analysis of H-NS binding in the genome by ChIP-Seq assays provided indirect evidence for the spreading of H-NS on DNA ''in vivo''. H-NS binds selectively to 458 regions in the genome. 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 (< 5 mM), H-NS would form rigid nucleoprotein filaments ''in vivo''. Alternatively, if there is an uneven distribution of magnesium in the cell, it could promote both DNA bridging and stiffening but in different regions of the nucleoid. Furthermore, H-NS is best known as a global gene silencer that preferentially inhibits transcription of horizontally transferred genes and it is the rigid filament that leads to gene silencing. Taken together, it appears that the formation of rigid filaments is the most likely outcome of H-NS-DNA interactions ''in vivo'' that leads to gene silencing but does not induce DNA condensation. Consistently, the absence of H-NS does not change the nucleoid volume. However, it is possible that ''E. coli'' experiences high-magnesium concentration under some environmental conditions. In such conditions, H-NS can switch from its filament inducing form to the bridge inducing form that contributes to DNA condensation and organization.Fis
Factor for Inversion Stimulation (Fis) is a sequence specific DNA binding protein that binds to specific DNA sequences containing a 15-bp symmetric motif. Like IHF, Fis induces DNA bending at cognate sites. The ability to bend DNA is apparent in the structure of Fis homodimer. A Fis homodimer possesses two helix-turn-helix (HTH) motifs, one from each monomer. An HTH motif typically recognizes the DNA major groove. However, the distance between the DNA recognition helices of the two HTH motifs in the Fis homodimer is 25 Å, that is ~ 8 Å shorter than the pitch of a canonical B-DNA, indicating that the protein must bend or twist DNA to bind stably. Consistently, theNucleoid-associated RNAs (naRNAs)
Early studies examining the effect of RNase A treatment on isolated nucleoids indicated that RNA participated in the stabilization of the nucleoid in the condensed state. 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. Most of the studies on HU focused on its DNA binding. However, HU also binds to dsRNA and RNA-DNA hybrids with a lower affinity similar to that with a linear dsDNA. 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. Several of them are non-coding RNAs, and one such RNA named naRNA4 (nucleoid-associated RNA 4), is encoded in a repetitive extragenic palindrome (''REP325''). In a strain lacking ''REP325'', the nucleoid is decondensed as it is in a strain lacking HU. naRNA4 most likely participate in DNA condensation by connecting DNA segments in the presence of 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.Supercoiling
Because of its helical structure, a double-stranded DNA molecule becomes topologically constrained in the covalently closed circular form which eliminates the rotation of the free ends. 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 causesPlectonemic supercoils in ''E. coli''
In most bacteria, DNA is present in supercoiled form. The circular nature of the ''E. coli'' chromosome makes it topologically constrained molecule that is mostly negatively supercoiled with an estimated average supercoiling density (σ) of -0.05. In the eukaryoticSources of supercoiling in ''E. coli''
Three factors contribute to generating and maintaining chromosomal DNA supercoiling in ''E. coli'': (i) activities of topoisomerases, (ii) the act of transcription, and (iii) NAPs.= Topoisomerases
= Topoisomerases are a particular category of DNA metabolic enzymes that create or remove supercoiling by breaking and then re-ligating DNA strands. ''E. coli'' possesses four topoisomerases.= 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.= Control of supercoiling by NAPs
= In the eukaryotic chromatin, DNA is rarely present in the free supercoiled form because nucleosomes restrain almost all negative supercoiling through tight binding of DNA to histones. Similarly, in ''E. coli'', nucleoprotein complexes formed by NAPs restrain half of the supercoiling density of the nucleoid. In other words, if a NAP dissociates from a nucleoprotein complex, the DNA would adopt the free, plectonemic form. DNA binding of HU, Fis, and H-NS has been experimentally shown to restrain negative supercoiling in a relaxed but topologically constrained DNA. 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. NAPs also regulate DNA supercoiling indirectly. Fis can modulate supercoiling by repressing the transcription of the genes encoding DNA gyrase. 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.Plectonemic supercoils organize into multiple topological domains
One of the striking features of the nucleoid is that plectonemic supercoils are organized into multiple topological domains. 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. Although identities of domain barriers remain to be established, possible mechanisms responsible for the formation of the barriers include: (i) A domain barrier could form when a protein with an ability to restrain supercoils simultaneously binds to two distinct sites on the chromosome forming a topologically isolated DNA loop or domain. It has been experimentally demonstrated that protein-mediated looping in supercoiled DNA can create a topological domain. 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
The nucleoid reorganizes in stationary phase cells suggesting that the nucleoid structure is highly dynamic, determined by the physiological state of cells. A comparison of high-resolution contact maps of the nucleoid revealed that the long-range contacts in the Ter macrodomain increased in the stationary phase, compared to the growth phase. Furthermore, CID boundaries in the stationary phase were different from those found in the growth phase. Finally, nucleoid morphology undergoes massive transformation during prolonged stationary phase; the nucleoid exhibits ordered, toroidal structures. Growth-phase specific changes in nucleoid structure could be brought about by a change in levels of nucleoid-associated DNA architectural proteins (the NAPs and the Muk subunits), supercoiling, and transcription activity. The abundance of NAPs and the Muk subunits changes according to the bacterial growth cycle. Fis and the starvation-induced DNA binding protein Dps, another NAP, are almost exclusively present in the growth phase and stationary phase respectively. Fis levels rise upon entry into exponential phase and then rapidly decline while cells are still in the exponential phase, reaching levels that are undetectable in stationary phase. While Fis levels start to decline, levels of Dps start to rise and reach a maximum in the stationary phase. A dramatic transition in the nucleoid structure observed in the prolonged stationary phase has been mainly attributed to Dps. It forms DNA/ crystalline assemblies that act to protect the nucleoid from DNA damaging agents present during starvation. HU, IHF, and H-NS are present in both growth phase and stationary phase. However, their abundance changes significantly such that HU and Fis are the most abundant NAPs in the growth phase, whereas IHF and Dps become the most abundant NAPs in the stationary phase. HUαα is the predominant form in early exponential phase, whereas the heterodimeric form predominates in the stationary phase, with minor amounts of homodimers. This transition has functional consequences regarding nucleoid structure, because the two forms appear to organize and condense DNA differently; both homo- and heterodimers form filaments, but only the homodimer can bring multiple DNA segments together to form a DNA network. The copy number of MukB increases two-fold in stationary phase. An increase in the number of MukB molecules could have influence on the processivity of the MukBEF complex as a DNA loop extruding factor resulting in larger or a greater number of the loops. Supercoiling can act in a concerted manner with DNA architectural proteins to reorganize the nucleoid. The overall supercoiling level decreases in the stationary phase, and supercoiling exhibits a different pattern at the regional level. Changes in supercoiling can alter the topological organization of the nucleoid. Furthermore, because a chromosomal region of high transcription activity forms a CID boundary, changes in transcription activity during different growth phases could alter the formation of CID boundaries, and thus the spatial organization of the nucleoid. It is possible that changes in CID boundaries observed in the stationary phase could be due to the high expression of a different set of genes in the stationary phase compared to the growth phase.Nucleoid structure and gene expression
NAPs and gene expression
The ''E. coli'' chromosome structure and gene expression appear to influence each other reciprocally. On the one hand, a correlation of a CID boundary with high transcription activity indicates that chromosome organization is driven by transcription. On the other hand, the 3D structure of DNA within nucleoid at every scale may be linked to gene expression. First, it has been shown that reorganization of the 3D architecture of the nucleoid in ''E. coli'' can dynamically modulate cellular transcription pattern. A mutant of HUa made the nucleoid very much condensed by increased positive superhelicity of the chromosomal DNA. Consequently, many genes were repressed, and many quiescent genes were expressed. Besides, there are many specific cases in which protein-mediated local architectural changes alter gene transcription. For example, the formation of rigid nucleoprotein filaments by H-NS blocks RNAP access to the promoter thus prevent gene transcription. Through gene silencing, H-NS acts as a global repressor preferentially inhibiting transcription of horizontally transferred genes. In another example, specific binding of HU at the ''gal'' operon facilitates the formation of a DNA loop that keeps the ''gal'' operon repressed in the absence of the inducer. The topologically distinct DNA micro-loop created by coherent bending of DNA by Fis at stable RNA promoters activates transcription. DNA bending by IHF differentially controls transcription from the two tandem promoters of the ''ilvGMEDA'' operon in ''E. coli''. Specific topological changes by NAPs not only regulate gene transcription, but are also involved in other processes such as DNA replication initiation, recombination, and transposition. In contrast to specific gene regulation, how higher-order chromosome structure and its dynamics influences gene expression globally at the molecular level remains to be worked out.DNA supercoiling and gene expression
A two-way interconnectedness exists between DNA supercoiling and gene transcription. Negative supercoiling of the promoter region can stimulate transcription by facilitating the promoter melting and by increasing the DNA binding affinity of a protein regulator. Stochastic bursts of transcription appear to be a general characteristic of highly expressed genes, and supercoiling levels of the DNA template contributes to transcriptional bursting. According to the twin supercoiling domain model, transcription of a gene can influence transcription of other nearby genes through a supercoiling relay. One such example is the activation of the ''leu-500'' promoter. Supercoiling not only mediates gene-specific changes, but it also mediates large-scale changes in gene expression. Topological organization of the nucleoid could allow independent expression of supercoiling-sensitive genes in different topological domains. A genome-scale map of unrestrained supercoiling showed that genomic regions have different steady-state supercoiling densities, indicating that the level of supercoiling differs in individual topological domains. As a result, a change in supercoiling can result in domain-specific gene expression, depending on the level of supercoiling in each domain. The effect of supercoiling on gene expression can be mediated by NAPs that directly or indirectly influence supercoiling. The effect of HU on gene expression appears to involve a change in supercoiling and perhaps a higher-order DNA organization. A positive correlation between DNA gyrase binding and upregulation of the genes caused by the absence of HU suggests that changes in supercoiling are responsible for differential expression. HU was also found to be responsible for a positional effect on gene expression by insulating transcriptional units by constraining transcription-induced supercoiling. Point mutations in HUa dramatically changed the gene expression profile of ''E. coli,'' altering its morphology,Spatial organization
Chromosomal interaction domains
In recent years, the advent of a molecular method called chromosome conformation capture (3C) has allowed studying a high-resolution spatial organization of chromosomes in both bacteria and eukaryotes. 3C and its version that is coupled withMacrodomains
Plectonemic DNA loops organized as topological domains or CIDs appear to coalesce further to form large spatially distinct domains called macrodomains (MDs). In ''E. coli,'' MDs were initially identified as large segments of the genome whose DNA markers localized together (co-localized) in fluorescence in situ hybridization (FISH) studies. A large genomic region (~1-Mb) covering '' oriC'' (origin of chromosome replication) locus co-localized and was called Ori macrodomain. Likewise, a large genomic region (~1-Mb) covering the replication terminus region (''ter'') co-localized and was called Ter macrodomain. MDs were later identified based on how frequently pairs of lambda ''att'' sites that were inserted at various distant locations in the chromosome recombined with each other. In this recombination-based method, a MD was defined as a large genomic region whose DNA sites can primarily recombine with each other, but not with those outside of that MD. The recombination-based method confirmed the Ori and Ter MDs that were identified in FISH studies and identified two additional MDs. The two additional MDs were formed by the additional ~1-Mb regions flanking the Ter and were referred to as Left and Right. These four MDs (Ori, Ter, Left, and Right) composed most of the genome, except for two genomic regions flanking the Ori. These two regions (NS-L and NS-R) were more flexible and non-structured compared to a MD as DNA sites in them recombined with DNA sites located in MDs on both sides. The genetic position of ''oriC'' appears to dictate the formation of MDs, because repositioning of ''oriC'' by genetic manipulation results in the reorganization of MDs. For example, genomic regions closest to the ''oriC'' always behave as an NS regardless of DNA sequence and regions further away always behave as MDs. The Hi-C technique further confirmed a hierarchical spatial organization of CIDs in the form of macrodomains. In other words, CIDs of a macrodomain physically interacted with each other more frequently than with CIDs of a neighboring macrodomain or with genomic loci outside of that macrodomain. The Hi-C data showed that the ''E. coli'' chromosome was partitioning into two distinct domains. The region surrounding ''ter'' formed an insulated domain that overlapped with the previously identified Ter MD. DNA-DNA contacts in this domain occurred only in the range of up to ~280 kb. The rest of the chromosome formed a single domain whose genomic loci exhibited contacts in the range of >280-kb. While most of the contacts in this domain were restricted to a maximum distance of ~500 kb, there were two loose regions whose genomic loci formed contacts at even greater distances (up to ~1 Mb). These loose regions corresponded to the previously identified flexible and less-structured regions (NS). The boundaries of the insulated domain encompassing ''ter'' and the two loose regions identified by the Hi-C method segmented the entire chromosome into six regions that correspond with the four MDs and two NS regions defined by recombination-based assays.Proteins that drive macrodomain formation
= MatP
= A search for protein(s) responsible for macrodomain formation led to identification of Macrodomain Ter protein (MatP). MatP almost exclusively binds in the Ter MD by recognizing a 13-bp motif called the macrodomain ''ter'' sequence (''matS''). There are 23 ''matS'' sites present in the Ter domain, on average there is one site every 35-kb. Further evidence of MatP binding in the Ter domain comes from fluorescence imaging of MatP. Discrete MatP foci were observed that co-localized with Ter domain DNA markers. A strong enrichment of ChIP-Seq signal in the Ter MD also corroborates the preferential binding of MatP to this domain. MatP condenses DNA in the Ter domain because the lack of MatP increased the distance between two fluorescent DNA markers located 100-kb apart in the Ter domain. Furthermore, MatP is a critical player in insulating the Ter domain from the rest of the chromosome. It promotes DNA-DNA contacts within the Ter domain but prevents contacts between the DNA loci of Ter domain and those of flanking regions. How does MatP condense DNA and promote DNA-DNA contacts? The experimental results are conflicting. MatP can form a DNA loop between two ''matS'' sites ''in vitro'' and its DNA looping activity depends on MatP tetramerization. Tetramerization occurs via coiled-coil interactions between two MatP molecules bound to DNA. One obvious model based on ''in vitro'' results is that MatP promotes DNA-DNA contacts ''in vivo'' by bridging ''matS'' sites. However, although MatP connected distant sites in Hi-C studies, it did not specifically connect the ''matS'' sites. Furthermore, a MatP mutant that was unable to form tetramers behaved like wild-type. These results argue against the ''matS'' bridging model for Ter organization, leaving the mechanism of MatP action elusive. One possibility is that MatP spreads to nearby DNA segments from its primary ''matS'' binding site and bridge distant sites via a mechanism that does not depend on the tetramerization.= MukBEF
= MukB belongs to a family of ATPases called structural maintenance of chromosome proteins (SMCs), which participate in higher-order chromosome organization in eukaryotes. Two MukB monomers associate via continuous antiparallel coiled-coil interaction forming a 100-nm long rigid rod. A flexible hinge region occurs in the middle of the rod. Due to the flexibility of the hinge region, MukB adopts a characteristic V-shape of the SMC family. The non-SMC subunits associating with MukB are MukE and MukF. The association closes the V formation, resulting in large ring-like structures. MukE and MukF are encoded together with MukB in the same operon in ''E. coli''. Deletion of either subunit results in the same phenotype suggesting that the MukBEF complex is the functional unit ''in vivo''. DNA binding activities of the complex reside in the MukB subunit, whereas MukE and MukF modulate MukB activity. MukBEF complex, together with Topo IV, is required for decatenation and repositioning of newly replicated ''oriC''s. The role of MukBEF is not restricted during DNA replication. It organizes and condenses DNA even in non-replicating cells. The recent high-resolution chromosome conformation map of the MukB-depleted ''E. coli'' strain reveals that MukB participates in the formation of DNA-DNA interactions on the entire chromosome, except in the Ter domain. How is MukB prevented from acting in the Ter domain? MatP physically interacts with MukB, thus preventing MukB from localizing to the Ter domain. This is evident in the DNA binding of MatP and MukB in the Ter domain. DNA binding of MatP is enriched in the Ter domain, whereas DNA binding of MukB is reduced compared to the rest of the genome. Furthermore, in a strain already lacking MatP, the absence of MukB causes a reduction in DNA contacts throughout the chromosome, including the Ter domain. This result agrees with the view that MatP displaces MukB from the Ter domain. How does the MukBEF complex function to organize the ''E. coli'' chromosome? According to the current view, SMC complexes organize chromosomes by extruding DNA loops. SMC complexes translocate along DNA to extrude loops in a cis-manner (on the same DNA molecule), wherein the size of loops depends on processivity of the complex. SMC complexes from different organisms differ in the mechanism of loop extrusion. Single molecule fluorescence microscopy of MukBEF in ''E. coli'' suggests that the minimum functional unit ''in vivo'' is a dimer of dimers. This unit is formed by joining of two ATP-bound MukBEF complexes through MukF-mediated dimerization. MukBEF localizes in the cell as 1-3 clusters that are elongated parallel to the long axis of the cell. Each cluster contains an average ~ 8-10 dimers of dimers. According to the current model, the MukBEF extrudes DNA loops in a “rock-climbing” manner. A dimer of the dimers releases one segment of DNA and capture a new DNA segment without dissociating from the chromosome. Besides DNA looping, a link between negative supercoiling and ''in vivo'' MukBEF function together with the ability of the MukB subunit to constrain negative supercoils ''in vitro'' suggests that MukBEF organizes DNA by generating supercoils.Role of NAPs and naRNAs
In addition to contributing to the chromosome compaction by bending, bridging, and looping DNA at a smaller scale (~1-kb), NAPs participate in DNA condensation and organization by promoting long-rang DNA-DNA contacts. Two NAPs, Fis and HU, emerged as the key players in promoting long-range DNA-DNA contacts that occur throughout the chromosome. It remains to be studied how DNA organization activities of Fis and HU that are well understood at a smaller scale (~1-kb) results in the formation of long-range DNA-DNA interactions. Nonetheless, some of the HU-mediated DNA interactions require the presence of naRNA4. naRNA4 also participates in making long-range DNA contacts. HU catalyzes some of the contacts, not all, suggesting that RNA participates with other NAPs in forming DNA contacts. HU also appears to act together with MukB to promote long-range DNA-DNA interactions. This view is based on observations that the absence of either HU or MukB caused a reduction in the same DNA-DNA contacts. It is unclear how MukB and HU potentially act together in promoting DNA-DNA interactions. It is possible that the two proteins interact physically. Alternatively, while MukBEF extrudes large DNA loops, HU condenses and organizes those loops.Role of functional relatedness of genes
There are reports that functionally-related genes of ''E. coli'' are physically together in 3-D space within the chromosome even though they are far apart by genetic distance. Spatial proximity of functionally-related genes not only make the biological functions more compartmentalized and efficient but would also contribute to the folding and spatial organization of the nucleoid. A recent study using fluorescent markers for detection of specific DNA loci examined pairwise physical distances between the seven rRNA operons that are genetically separated from each other (by as much as two million bp). It reported that all of the operons, except ''rrn''C, were in physical proximity. Surprisingly, 3C-seq studies did not reveal the physical clustering of ''rrn'' operons, contradicting the results of the fluorescence-based study. Therefore, further investigation is required to resolve these contradicting observations. In another example, GalR, forms an interaction network of GalR binding sites that are scattered across the chromosome. GalR is a transcriptional regulator of the galactose regulon composed of genes encoding enzymes for transport and metabolism of the sugar D-galactose. GalR exists in only one to two foci in cells and can self-assemble into large ordered structures. Therefore, it appears that DNA-bound GalR multimerizes to form long-distance interactions.Global shape and structure
ConventionalNucleoid-membrane connections
An expansion force due to DNA-membrane connections appears to function in opposition to condensation forces to maintain an optimal condensation level of the nucleoid. Cell-fractionation and electron microscopy studies first indicated the possibility of DNA-membrane connections. There are now several known examples of DNA-membrane connections. Transertion is a mechanism of concurrent transcription, translation, and insertion of nascent membrane proteins that forms transient DNA-membrane contacts. Transertion of two membrane proteins LacY and TetA has been demonstrated to cause the repositioning of chromosomal loci toward the membrane. Another mechanism of nucleoid-membrane connections is through a direct contact between membrane-anchored transcription regulators and their target sites in the chromosome. One example of such as transcription regulator in ''E. coli'' is CadC. CadC contains a periplasmic sensory domain and a cytoplasmic DNA binding domain. Sensing of an acidic environment by its periplasmic sensory domain stimulates DNA binding activity of CadC, which then activates transcription of its target genes. The membrane-localization of genes regulated by a membrane-anchored transcription regulator is yet to be demonstrated. Nonetheless, activation of target genes in the chromosome by these regulators is expected to result in a nucleoid-membrane contact albeit it would be a dynamic contact. Besides these examples, the chromosome is also specifically anchored to the cell membrane through protein-protein interaction between DNA-bound proteins, e.g., SlmA and MatP, and the divisome. Since membrane-protein encoding genes are distributed throughout the genome, dynamic DNA-membrane contacts through transertion can act as a nucleoid expansion force. This expansion force would function in opposition to condensation forces to maintain an optimal condensation level. The formation of highly condensed nucleoids upon the exposure of ''E. coli'' cells to chloramphenicol, which blocks translation, provides support for the expansion force of transient DNA-membrane contacts formed through transertion. The round shape of overly-condensed nucleoids after chloramphenicol treatment also suggests a role for transertion-mediated DNA-membrane contacts in defining the ellipsoid shape of the nucleoid.Visualization
The nucleoid can be clearly visualized on an electron micrograph at very highDNA damage and repair
Changes in the structure of the nucleoid of bacteria and archaea are observed after exposure to DNA damaging conditions. The nucleoids of the bacteria ''Bacillus subtilis'' and ''Escherichia coli'' both become significantly more compact after UV irradiation. Formation of the compact structure in ''E. coli'' requires RecA activation through specific RecA-DNA interactions. The RecA protein plays a key role in homologous recombinational repair of DNA damage. Similar to ''B. subtilis'' and ''E. coli'' above, exposures of the archaeon ''Haloferax volcanii'' to stresses that damage DNA cause compaction and reorganization of the nucleoid. Compaction depends on the Mre11-Rad50 protein complex that catalyzes an early step in homologous recombinational repair of double-strand breaks in DNA. It has been proposed that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate targets, and by facilitating the search for intact DNA sequences during homologous recombination.See also
*References
{{Organelles Bacteriology Cell anatomy