Nucleoid: Definition, Structure, Dynamics and Function of This Containing Region of DNA

It is the region that contains DNA in prokaryotes.

Microorganisms have provided powerful systems for understanding the basic principles of chromosome organization, dynamics, and function, beginning with the replicon hypothesis .

Over the past decade, increasingly sophisticated image analysis from physical perspectives has been added to the more traditional tools provided by biochemistry, genetics, and molecular biology.

A major challenge for current research is integrating all of these considerations to provide a more in-depth and unified view.

The nucleoid

The nucleoid is the space within a prokaryotic cell where genetic information is located, called a genophore. Prokaryotes are divided into bacteria and archaea, which are single-celled organisms that do not contain membrane-bound organelles.

The nucleoid, then, does not have a membrane around it either. It adheres to the cell membrane and is in immediate contact with the cytoplasm.

The nucleoid is also not uniform in shape and does not have a specific size. However, we can still distinguish it from the rest of the cell and identify it under a light microscope.

The nucleoid is mainly composed of multiple copies packed into a continuous strand of DNA, with the addition of some RNA and proteins. DNA in prokaryotes is double-stranded and generally takes a circular shape.

Note that DNA can sometimes be found in other regions outside of the nucleoid as well. To put things in perspective, we can look at the eukaryotic counterpart of the nucleoid.

Eukaryotes, like plants and animals, have a nucleus that houses their genetic material, with a surrounding double membrane, or what we call the nuclear envelope.

This membrane separates the contents of the nucleus from the cytoplasm. As in prokaryotes, eukaryotic DNA is also double-stranded.

In bacteria

An important advance in recent years has been the appreciation that the circular DNA of the bacterial chromosome, called “nucleoid”, is a discrete and well-defined physical object.

Fresh fluorescence images of living cells and fixed cell chromosome capture analysis reveal discrete shapes with defined longitudinal substructure.

These findings confirm and extend previous image analysis findings and put models in which the nucleoid comprises a randomly oriented fiber, either linear or topologically branched, that fills the available cell space to rest.

Nucleosides are prepared from isolated plastids by solubilizing the envelope and thylakoid membranes. Various nonionic detergents have been used, as well as various density gradient media.

However, the most important improvement was the use of the so-called “TAN” tampon invented by the Kuroiwa group. “TAN” was the name of a researcher who first used it in his laboratory.

The TAN buffer consists of 0.5 M sucrose, 1.2 mM spermidine, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride, and 20 mM trisaminomethane hydrochloride (Tris HCL) (pH 7.5). Sucrose and spermidine are highly effective in stabilizing the compact structure of nucleoids.

Low ionic strength and chelation of divalent cations with ethylenediaminetetraacetic acid are essential to maintain nucleoids in a highly condensed state.

The addition of sodium chloride (NaCl) at a concentration above 0.2 M or several millimolar magnesium chloride (MgCl2) releases major DNA-binding proteins from nucleosides.

Spermidine is a member of natural polyamines, which are present in chloroplasts as well as in bacterial or eukaryotic cells and are important for maintaining proper gene expression activity.

However, spermidine should not be added to the buffer during isolation of plastids by the Percoll density gradient, as it binds to the negatively charged surface of Percoll.

A simple and convenient method for preparing nucleoids from plastids is to:

Solubilization of plastids with Nonidet P-40, low speed centrifugation, and filtration through a membrane filter to remove bulk contaminants (starch, cell nucleus fragments, cuticles, and other insoluble materials), and nucleoid sedimentation by high speed centrifugation.

The use of density gradient centrifugation through metrizamide or sucrose is effective in purifying nucleoids to some extent, but the effectiveness of the density gradient is limited by the fact that nucleoids are highly heterogeneous with respect to buoyancy density.

Unfortunately, even the purest of currently available plastid preparations contain several hydrophobic proteins that are not believed to be true constituents of nucleosides.

Although isolated nucleoid preparations appear very pure upon inspection under a fluorescence microscope after staining with fluorochrome DAPI (4 ‘, 6-diamidino-2-phenylindole), several hydrophobic proteins originating from various cell membranes adhere to the preparation. nucleoid.

As demonstrated by the microsequencing of representative protein bands of the nucleoids.

This may be one reason why a comprehensive proteomic analysis of plastid nucleosides has not been performed. Currently, the important components are identified one by one by molecular biological and biochemical means.

Various DNA-binding proteins are identified by “reconstitution” experiments in which a protein overexpressed in Escherichia coli cells and highly purified from them is mixed with the purified DNA to see the formation of particulate structures that resemble cells. nucleosides under microscopy.

Another method is to use DAPI as a fluorescent probe to monitor DNA condensation. This method is based on changes in fluorescence quenching caused by condensation of DNA and fluorochrome.

The effect of the addition of nucleoid proteins, which are overexpressed in E. coli cells, on the transcriptional activity of nucleoids is a good criterion for identifying functionally important components of nucleoids, an approach that complements biochemical analysis.

The protein composition of nucleoids isolated from several different plants and organs has been reported.

Changes in protein composition or transcription activity during development of chloroplasts, chromoplasts, proplastides, and amyloplasts are described.

The nucleosides of etioplasts and chloroplasts in cucumbers and peas have been compared.

The protein composition is mostly identical in cucumber cotyledon etioplasts and chloroplasts, while the pea leaf etioplast and chloroplast nucleoplasts contained qualitatively and quantitatively different proteins.

The etioplasts of cucumber cotyledons are large and contain highly developed prolamellar bodies, ready to become chloroplasts shortly after the onset of illumination (about 3 to 6 h).

While the etioplasts of pea leaves are small and begin to develop for a long time (more than 24 h) after the onset of illumination.

This suggests that the composition of the nucleoids is determined not only by the state of greening, but by the state of global development of the plastid.

Nuclear structure and chromosome replication

The central feature of bacterial nucleic acid metabolism is the nucleoid, a cytoplasmic region that is so densely packed with nuclear material that it excludes ribosomes and can be easily seen on electron micrographs.

The nucleoid generally occupies about 10% of the cell volume. The nucleoid is not enclosed by a nuclear membrane as is found in eukaryotes, which has significant consequences for the organism.

The nucleoid is made up of approximately 60% DNA, 30% RNA, and 10% proteins. The chromosome, made up of DNA, is the predominant molecule in the nucleoid.

Typically, there is a chromosome approximately 1 mm in length, with a typical prokaryotic genome size on the order of 1.9 to 6.3 x 106 base pairs.

This DNA is a double-stranded, circular, covalently closed (usually) supercoiled molecule.

There are several species of prokaryotic microorganisms that have linear chromosomes, and some species have two or three chromosomes.

Considering the large size of the chromosome, it is not surprising that it must be compacted to fit in the bacterial cell. Since there is no nuclear membrane, RNA and protein keep the nuclear structure compact.

The chromosome exists as 50–100 separately coiled domains that can be individually relaxed for gene replication, repair, and expression.

Supercoiling is maintained by topoisomerase enzymes, such as DNA gyrase (maintains negative supercoiling and relaxes).

Other proteins important for maintaining nucleoid structure are the non-specific HU DNA-binding proteins and histone-like nucleoleum structuring (H-NS), and the IHF (host integrating factor) protein which has sequence binding sites of Specific DNA.

Chromosome replication occurs by the same universal principles that guide replication in all biological systems.

That is, DNA synthesis is semi-conservative and always occurs in the 5 ′ to 3 ′ direction.

DNA synthesis is semi-continuous, and DNA polymerase requires a template and a primer.

In most bacteria, there are three DNA polymerase enzymes, with the holoenzyme DNA polymerase III complex being responsible for chromosome replication.

DNA synthesis always begins at the same site called the origin of replication and is bidirectional from that origin.

In addition to the polymerase complex, there are a variety of other enzymes that contribute to initiation and synthesis. Some of these enzymes can be targets for antibiotics.

The nucleoid is self-adhesive

Several lines of evidence reveal that there is a strong tendency to general coalescence of chromosomal material, that is, that the nucleoid is “self-adherent”.

The images reveal that virtually all of the chromosomal DNA is part of the nucleoid shape.

Furthermore, during the replication / segregation process, it can sometimes be observed that elongation involves lobes of the newly replicated material protruding, implying an intrinsic dynamic tendency for coalescence in elongated shapes.

Finally, self-adherence is implicit in the finding that individual loci and pairs of loci tend to have fairly fixed positions relative to each other in the resting nucleosides (G1).

Radial, but not longitudinal, confinement

Non-septate cells exhibit discrete nucleoside chains in the absence of intercellular boundaries; furthermore, the G1 nucleoid does not always extend to the end of the cell.

Therefore, the nucleoid is a discrete object in the absence of “longitudinal confinement.”

In contrast, the nucleoid touches the inner periphery of the cell in the radial dimension.

Since the shape tends to be helically curved, this contact is not uniform but rather reflects the helical path.

One implication of this configuration is that the nucleoid tends to define a complementary helical space around the periphery of the cell.

Regardless of the molecular linkages between the nucleoid and the inner cell membrane, it also appears that the nucleoid as a whole tends to “push” the periphery of the cell out, that is, that the shape is “radially confined.”

The confinement of the nucleoid in the radial dimension figures prominently in various aspects of the organization, arrangement, and function of chromosomes.

At “G1” a curved ellipsoidal shape with underlying longitudinal duality

The nucleoid before replication (“G1”), as defined in E.coli and C. crescentus, tends to be thinner at the ends than in the middle, that is, be ellipsoidal and also deform into a helix with a smooth curve.

The curvature does not necessarily have a specific ability, which implies that the important characteristic is the curvature itself. Furthermore, nucleoid DNA is more dense centrally than radially.

Behind this overall shape is the fact that DNA tends to organize itself into a pair of parallel beams that extend longitudinally along the length of the nucleoid and rotate smoothly relative to each other to give the nucleoid shape of a helical, smoothly curved shape. .

In C. crescentus, the two packages correspond to the left and right replichores.

In some circumstances, including late stages of the cell cycle, the nucleoid appears as an open ring and / or well-separated bundle pairs.

Longitudinal confinement could potentially influence form at these stages.

Dual Radial Arrays of Plectonemic Loops

Chromosome capture analysis in C. crescentus suggests that, in this organism, the longitudinal duality probably reflects the existence of two parallel objects arranged in a ‘bottle brush’ shape, each comprising a radial series of plectonemic loops. .

Each loop would be ~ 15kb long with a ~ 100kb super arrangement. A similar underlying organization likely explains the duality in E.coli.

If so, the two characteristics identified could reconcile previous observations in E.coli that differently defined a topologically supercoiled domain by ~ 50–100kb versus domains of ~ 10–15kb).

Domain differentiation

For most Enterobacteriaceae and Vibrionaceae, the terminal region is highly structured through a dedicated protein, MatP, a plectonema-binding protein with a preferred binding sequence whose domain location is specifically nucleated.

The origin is also embedded in a structured region as shown in B.subtils and E.coli. In B. subtili this structure is created by condensin and nucleated by ParB. The origin and termination domains also occur in P.aeruginosa.

For E.coli , domain differentiation of interstitial regions has also been suggested.

The experimental definitions of these regions can reflect both the segregation process and the physical transitions of different macrosegments of the genome.

Dynamic

The time-lapse images have defined local dynamic movements and have also revealed global dynamic behaviors.

Movement of loci on short time scales

The local movement of a fluorescently labeled chromosomal locus is defined by plotting its mean square displacement (DCM) versus elapsed time of observation.

In a log-log plot, simple diffusive motion provides a linear relationship with a slope of 1.

For the chromosomal loci in bacteria, the relationship is linear but with a slope of less than 1, which implies a “sub-diffusive” movement.

This behavior is due to the viscoelastic nature of the crowded nucleoid: as a locus moves, it pushes against the constraints of the environment, which push back, thus preventing movement.

The alternative explanations, that is, the adherence of the place to the characteristics found or impediments of the fixed obstacles, are not compatible with the observed patterns.

An important subcomponent of the apparent diffusion coefficient is non-thermal in nature, with ATP-dependent processes mechanically promoting agitation.

So far, no single dominant motor protein has been identified, with DNA gyrase, cell wall biosynthesis, and the MreB cytoskeleton excluded as major players and RNA polymerase having only a minor role.

A monitored locus may occasionally exhibit rapid movements exhibiting “near ballistic” dynamics, involving active translocation machinery or stress-relaxation effects.

Such movements occur for loci throughout the genome, but differently with different genomic coordinates.

These movements do not strictly correlate with DNA replication, they tend to occur along the length of the nucleoid, and sometimes accompany chromosome segregation transitions.

Two types of global dynamics at the nucleoside level

Whole nucleoid images in E. coli reveal two dynamic behaviors that involve the entire nucleoid.

These two behaviors come into play on different time scales. In no case is the underlying mechanism known.

In both cases, the elimination of tethers between segments (mediated by proteins and / or topological, lengthwise and between sisters) is proposed to perform critical functions, making the nucleoid more “fluid”.

Fluency would facilitate the local movements required for various chromosomal processes, including, for example, the displacement of transcribed regions to move to the nuclear periphery for translation, as well as the dynamics of replication, sister segregation, and organization.

Longitudinal density waves

The total nucleoside density fluctuates along the length of the nucleoid with a periodicity of one to two minutes, probably throughout the cell cycle, with a net displacement of approximately 5% of the nucleoside material in every 5 s .

These waves are proposed to promote the internal mobility of the nucleoid by promoting the loss of ties or entanglements between segments that would otherwise create a gel.

Such a role would be analogous to that suspected for the back and forth movements of meiotic prophase chromosomes in correlation with the removal of unwanted tangles created during chromosome pairing.

Cyclic nucleoid extension and shortening

The length of the cell increases monotonically during growth. In contrast, the length of the nucleosides varies discontinuously, in a cyclic pattern.

In each cycle, a five-minute period of nucleoid shortening is followed by a 20-minute period of lengthening.

The rate of elongation increases from ~ 10min to a maximum and then decreases, finally becoming negative as shortening occurs, followed by the next phase of elongation.

These kinetics are surprisingly consonant with the accumulation, release and dissipation of viscoelastic mechanical stress, which implies the existence of mechanical stress cycles.

So far, these cycles are documented for a period that includes, but extends well beyond the period of DNA replication, with indications that it also occurs in G1.

Such cycles could comprise a primordial cell cycle that governs the program of chromosomal events and its link to the cycle of cell division.

These global cycles are also specifically implicated in sister segregation.

It is proposed that random tethers between segments prevent the formation of a regular nucleoleum organization (e.g. radial loop arrays), which tends to make the nucleoid larger and less elongated (i.e. wider and shorter ) in a high energy state (mechanical stress).

Eventually, the stress level will become high enough to cause release of the restraints, thus allowing stress relief from lengthening.

This effect would be concomitantly blocked by the development of the organization (radial loop).

At first glance, it may seem counterintuitive that the expansion provided by nucleoide dynamics could be biologically useful, as it is commonly accepted that chromosome compaction is a key element in chromosome segregation and organization.

However, several scenarios can be imagined in which transient decay could help.

For example, one must remember that global condensation will not separate sisters and that specifically only condensation of length is capable of driving segregation.

A general proposition is that such sister-specific condensation (generated, for example, by supercoiling) will be affected or at least slowed down by molecular cross-links.

Therefore, a local nucleoid expansion could decrease the amount of cross-links, subsequently allowing a more efficient longitudinal condensation.

More generally, there is general agreement that the underlying organization of DNA within the nucleoid is relatively regular.

Molecular events, and in particular transcription, will disrupt that organization, and those disruptions can be restricted and / or permanent by molecular cross-links.

Transient expansion waves could once again suppress these crosslinks, allowing a reorganization of the nucleoid in its “ideal” state.

Nucleoid function

The nucleoid is essential to control cell activity and reproduction. It is where DNA transcription and replication take place.

Within it, we can expect to find enzymes that serve as biological catalysts and help with replication, as well as other proteins that have other functional and structural roles, such as helping DNA formation, facilitating cell growth, and regulating the genetic material of the cell. .

Conclution

The dynamics of prokaryotic and eukaryotic chromosomes have always been predicted to be fundamentally similar in some way.

Recent observations provide strong support for this proposition and clarify the nature of the relationships.

Chromosomes tend to be discrete and coherent units in bacteria as is well known for eukarotes.

The elastic environment that governs short-term locus mobility and its dependence on ATP (enzyme) is similar in both cell types.

So is the observation that individual loci undergo ballistic movements. Chromosomes have a fundamental underlying organization that involves a radial series of loops, with higher-order organization tracks.

Furthermore, in both cases, the central “backbone” defined by the loop bases and associated proteins includes condensin, AT-rich sequences, and architectural proteins that bind to these sequences.

The back and forth oscillations of the longitudinal density waves observed in E. coli have direct parallels in mammalian G1 nuclei as well as in meiotic prophase.

The global cycles of contraction / extension observed in E. coli appear to be very parallel to the global cycles of chromosome expansion / contraction observed in the meiotic and mitotic programs of the eukaryotic programs, which are also related to the individualization of the chromatid period. sisters.

Sister segregation at anaphase in eukaryotes is preceded by morphological individualization of the sisters before and independently of spindle forces, in accordance with emerging evidence that global internal factors play a central role for the individualization of sisters in the bacteria.