SBC 312 Genome Organizatio II(1)

7/31/2019 SBC 312 Genome Organizatio II(1)

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SBC: 312 Genome Organizations II N

Kenyatta UniversityCourse outline: Higher order structure in chromatin, organization of chromatin in thecell nucleus, Heterochromatin and Euchromatin, Scaffolds and Dormains. The nuclearmatrix, The boundary of eukaryotic Nucleus: The nuclear envelope structure, Transportthrough the nuclear envelop, Nuclear structure in Prokaryotes, bacterial nucleoidstructure, bacterial nucleiod proteins, Nucleiod Structure in Cyanobacteria,Dinoflagellates; Eukaryotes with Prokaryotic DNA organization

Reference: Genes VIII Benjamin Lewin, Genetics. Analysis of genes and genomes 5thEd.Daniel L. Hartl and Elizabeth W. Jones

Introduction:

1870, it was observed that the nucleus plays a key role in inheritance. Soon therefore,chromosomes were first observed inside nucleus as threadlike objects and that whereas thenumber of chromosomes in each cell differs among biological species, the number ofchromosomes in nearly always constant within the cell of any particular species

The genome is the complete set of sequences in the genetic material of an organism. It includesthe sequence of each chromosome plus any DNA in organelles. In sexual organisms, the genomeis usually regarded as the DNA present in a reproductive cell. The human genome, which iscontained in the chromosomes of a sperm or egg, includes approximately 3 billion nucleotidepairs of DNA. The complete set of proteins encoded in the genome is known as the proteome.The study of genomes constitutes genomics; the study of proteomes constitutes proteomics. Thefundamental unity of life can be seen in the similarity of proteins in the proteomes of diversetypes of organisms..

The structure of eukaryotic chromosomeRemarkable feature of genetic apparatus is the feat of packaging where enormous length of geneticmaterial is condensed into a relatively few small chromosomes. The largest of the 23 humanchromosomes contain a DNA molecule that is 82 mm in length. However, at metaphase of mitoticdivision, this DNA is condensed into compact structure about 10m in diameter. For instance, if aDNA molecule in human chromosome one, the largest were a cooked spaghetti noodle 1mm indiameter, it would stretch for 25 miles; in chromosome condensation, this noodle is gathered together,coil upon coil, until at metaphase it as acanoe sized tangle of spaghetti 16 feet long and 2 feet wide.After cell division the noodle is unwound again.


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Molecular GeneticsChromosome

DNA

Nucleotides

Nucleus

Cell

The Nucleosome-The structural Unit of chromatin.DNA of eukaryotic chromosome is associated with numerous protein molecule in a stable, orderedaggregate called chromatin. Some of the proteins present in chromatin determine chromosomestructure and the changes in structure that occur during the cell division cycle. Other chromatinproteins play important roles in regulating chromosome functions.

Genetic Concepts

Chromosome

double stranded DNA

molecule packaged by

histone & scaffold

proteins

DNA double helix

nucleosome

30nm fiber

condensed chromosome


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The Nucleosome core particle

Simplest form of chromatin occurs in non-dividing cells, when chromosomes are not tightlycondensed. Chromatin of such cells is a complex aggregate of DNA and protein. The major class ofproteins comprises the histoneproteins, which are largely responsible for the structure of chromatin.Five major types of histones- H1,H2A,H2B,H3 and H4- are present in chromatin of nearly alleukaryotic cells in amounts about equal in mass to the of DNA.

Histones are small proteins (100-200 amino acids) differing from other proteins in that from 20-30 %of the amino acids are lysine and arginine, both positive amino acids. (only a few % of the aminoacids of a typical protein are lysine and arginine). The positive charges enable histone to bind DNAwhich is negatively charged, primarily by electrostatic attraction to the negatively charged phosphategroups in a sugar-phosphate back-bone of DNA. Chromatin placed in solution of high saltconcentration eg 2 molar NaCl, to eliminate electrostatic attraction causes histones to dissociate fromDNA. Histones also bind tightly to each other; both DNA-Histone and Histone-histone binding areimportant for chromatin structure.

Histones from different organisms are similar, with exception of H1. Infact the amino acid sequenceof H3 from widely different species are almost identical. Eg. A cows H3 and Pea H3 differ by only 4of the 135 amino acids. H4 of all organisms also are quite similar, cow and pea H4 differ by only 2 ofthe 102 amino acids. There are few proteins whose amino acid sequences vary so little from onespecies to the next. When this variation is between organism is very small, it is said that the sequenceis highly conserved. The exra-ordinary conservation of histone composition over the millions ofyears of evolutionary divergence is consistent with the important role these proteins play in thestructural organization of eukaryotic chromosomes

Chromatin resembles a regularly beaded thread figure 8.6. This bead like unit is called nucleosome.The organization of nucleosome is illustrated in figure 8.7. Part A; Each unit has has a definitecomposition, consisting of two molecules of H2A,H2B,H3 and H4, a segment of DNA containingabout 200 nucleotides and one H1. The complex of two subunits each of H2A,H2B,H3 and H4 as well

as part of the DNA forms a 'bead' and the remaining DNA and H1 bridges between thebeads.Treatment of chromatin with DNAse (eg Micrococcla nuclease from bacterium Staphylociccusaureus ) yields a collection of small particles of quite uniform size consisting only of histones andDNA. The DNA fragments in these particles are of lengths equal to about 200 nucleotides or smallmultiple of that unit size (the precise size varies with species and tissues). These particles results fromcleavage of the linker DNA segments between the beadsfigure 8.7b. Treatment with DNAse results inloss of H1 and digestion of all the DNA except that protected by histones in the bead. The resultingstructure is called a core particle, figure 8.8. It consists of two molecules of each H2a,H2B,H3 andH4 around which is wound a segment of DNA of about 145 nucleotides. Each nucleosome is


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composed of a core particle,, linker DNA between the adjacent core particles (which is removed byextensive DNAse activity), and one H1, the H1 binds to the histone octamer and to the linker DNA,causing the linker that extend from both sides of the core particle to cross and draw nearer to theoctamer, though some of the linker DNA do not come into contact with any histone. Linker sizeranges from 20-100 nucleostides from different organisms and even cell types in the same organism(55 nucleotides is usually considered an average size).

One molecule of H1 binds to the site at which DNA enters and leaves each nucleosome, and achain of H1 molecules coils the string of nucleosomes into the solenoid structure of the chromatinfibre. Nucleosomes not only neutralize the charges of DNA, but they have other consequences.First, they are an efficient means of packaging. DNA becomes compacted by a factor of six whenwound into nucleosomes and by a factor of about 40 when the nucleosomes are coiled into asolenoid chromatin fibre. The winding into nucleosomes also allows some inactive DNA to befolded away in inaccessible conformations, a process that contributes to the selectivity of geneexpression.Arrangement of chromatin fibre in a chromosome

The chromosomal DNA is folded and refolded again such that it is convenient to think of chromosomeas having several hierarchical levels of organization, each responsible for a particular degree ofshortening of enormous DNA figure 8.9 assemble of DNA and Histones can be considered the firstlevel of organization- namely, a sevenfold reduction in length of DNA and formation of a beadedflexible fibre 110 widefigure 8.9b., roughly five times the width of free DNAfigure 8.9a . structureof chromatin differs with salt concentration, and the 110 fibre is only present when saltconcentration is low. If the concentration is increased slightly, the fibre becomes shortened somewhatforming a zigzag arrangement of closely spaced beads between which the linking DNA is no longervisible in electron micrographs.If salt concentration is further increased to that presnet in living cells, then the second level ofcompaction occurs, the organization of the 110 nucleosome fibre into a shorter, thicker fibre with anaverage diameter ranging from 300-330, it is called the 30 nm fibre. In forming this structure, the110 fibre apparently coils in a somewhat irregular left-handed superhelix or solenoidal supercoilwith six nucleosomes per turn figure 8.10. Most intracellular chromatin is believed to have the

solenoidal supercoiled configuration

The final level of organization is that in which the 30nm fibre condenses into a chromatid of compactmetaphase chromosome figure 8.9d through f. little information is known about the additional foldingthat is required of the fibre in each loops of DNA to produce the fully re-condensed metaphasechromosome. The genetic significance of the compaction of DNA and protein into chromatin andultimately into chromosome is that it greatly facilitates the movement of the genetic material duringnuclear division. Relative to a fully extended DNA molecule, the length of the extended metaphasechromosome is reduced by a factor of approximately 10000 times as a result, chromosomecondensation, without which, chromosomes would become so entangled that there would be manymore abnormalities in the distribution of genetic material into daughter cells.


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Organization of Chromatin Fibre

Several studies indicate that chromatin is organized into a series of large radial loops anchored tospecific scaffold proteins. Each loop consists of a chain of nucleosomes and may be related tounits of genetic organization. This radial arrangement of chromatin loops compacts DNA about athousand fold. Further compaction is achieved by a coiling of the entire looped chromatin fibreinto a dense structure called a chromatid, two of which form the chromosome. During celldivision, this coiling produces a 10,000-fold compaction of DNA.


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4-12Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Anatomy of a chromosomeAnatomy of a chromosome

Metaphase chromosomes are classified by the position of the centromere

Fig. 4.3

4-15Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Autosomes pairs of nonsex chromosomes

Sex chromosomes and autosomes are arranged in homologous pairs

Note 22 pairs of autosomes and 1 pair of sex chromosomes


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Euchromatin versus Heterochromatin

The density of the chromatin that makes up each chromosome (that is, how tightly it is packed)varies along the length of the chromosome.

dense regions are called heterochromatin less dense regions are called euchromatin.

Heterochromatin

is found in parts of the chromosome where there are few or no genes, such aso centromeres ando telomeres

is densely-packed; is greatly enriched with transposons and other "junk" DNA; is replicated late in S phase of the cell cycle has reduced crossing over in meiosis Those genes present in heterochromatin are generally inactive; that is, not transcribed and

show increased methylation of the cytosines in CpG islands of the gene's promoter(s) The histones in the nucleosomes of heterochromatin show characteristic modifications:

o decreased acetylation;


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o increased methylation of lysine-9 in histone H3 (H3K9),which now provides a binding site forheterochromatinprotein 1 (HP1), which blocks access by the transcriptionfactors needed for gene transcription.

o increase methylation of lysine-27 in histone H3 (H3K27).

Euchromatin

is found in parts of the chromosome that contain many genes; is loosely-packed in loops of30-nm fibers. These are separated from adjacent heterochromatin by insulators.

In yeast, the loops are often found near the nuclear pore complexes. However, in animalcells, active gene transcription occurs near the center of the nucleus and appears to berepressed (heterochromatin) near the inner surface of the nuclear envelope.

The genes in euchromatin are active and showo decreased methylation of the cytosines in CpG islands of the gene's promoter(s);o increased acetylation of nearby histones ando decreased methylation of lysine-9 and lysine-27 in histone H3.

The diagram represents a hypothetical model of how euchromatin and heterochromatin may beorganized during interphase in a yeast cell (but not in an animal cell).

Histone Modifications

Although their amino acid sequence (primary structure) is unvarying, individual histonemolecules do vary in structure as a result of chemical modifications that occur later to

individual amino acids.

These include adding:

acetyl groups (CH3CO) to lysines phosphate groups to serines and threonines methyl groups to lysines and arginines

Although 7580% of the histone molecule is incorporated in the core, the remainder attheN-terminal dangles out from the core as a "tail" (not shown in the figure).

The chemical modifications occur on these tails, especially of H3 and H4. Most of theseschanges are reversible. For example, acetyl groups are

addedby enzymes called histone acetyltransferases (HATs)(not to be confusedwith the "HAT" medium used to make monoclonal antibodies!) and

removedby histone deacetylases (HDACs).


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More often than not, acetylation of histone tails occurs in regions of chromatin thatbecome active in gene transcription. This makes a kind of intuitive sense as adding acetylgroups neutralizes the positive charges on Lys thus reducing the strength of theassociation between the highly-negative DNA and the highly-positive histones.

But there is surely more to the story.

Acetylation of Lys-16 on H4 ("H4K16ac") prevents the interaction of their "tails"needed to form the compact 30-nm structure of inactive chromatin and thus isassociated with active genes. Note that this case involves interrupting protein-protein not protein-DNA interactions.

Methylation, which also neutralizes the charge on lysines (and arginines), caneither stimulate orinhibit gene transcription in that region.

o Methylation of lysine-4 in H3 ("H3K4me") is associated with active geneswhile

o methylation of lysine-9 and/or lysine-27 in H3 (H3K9me and H3K27me

respectively) is associated with inactive genes. (These include thoseimprinted genes that have been permanently inactivated in somaticcells. Link to discussion.)

And adding phosphates causes the chromosomes to become more not less compact as they get ready for mitosis and meiosis.

In any case, it is now clear that histones are a dynamic component of chromatin and notsimply inert DNA-packing material.

Nucleosomes and Transcription

Transcription factors cannot bind to theirpromoterif the promoter is blocked by anucleosome. One of the first functions of the assembling transcription factors is to eitherexpel the nucleosome from the site where transcription begins or at least to slide thenucleosomes along the DNA molecule. Either action exposes the gene's promoter so thatthe transcription factors can then bind to it.

The actual transcription of protein-coding genes is done by RNA polymerase II (RNAPII). In order for it to travel along the DNA to be transcribed, a complex of proteinsremoves the nucleosomes in front of it and then replaces them after RNAP II hastranscribed that portion of DNA and moved on.

The Nuclear Envelope

The nucleus, the largest organelle in animal cells, is surrounded by two membranes, each one aphospholipid bilayer containing many different types of proteins. The inner nuclear membrane defines thenucleus itself. In most cells, the outer nuclear membrane is continuous with the rough endoplasmicreticulum, and the space between the inner and outer nuclear membranes is continuous with the lumen of


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the rough endoplasmic reticulum. The space between the outer and inner membranes is alsocontinuous with rough endoplasmic reticulum space. It can fill with newly synthesizedproteins just as the rough endoplasmic reticulum does. The nuclear envelope is enmeshedin a network of filaments for stability.

The inner surface of the nuclear envelope has a protein liningcalled the nuclear lamina, which binds to chromatin and other contents of the nucleus. Figurebelow The two nuclear membranes appear to fuse at nuclear pores, the ringlike complexes composed ofspecific membrane proteins through which material moves between the nucleus and the cytosol. Thestructure of nuclear pores and the regulated transport of material through them are detailed in Chapter 12The entire envelope is perforated by numerous nuclear pores. These transport routes are fullypermeable to small molecules up to the size of the smallest proteins, but they form a selectivebarrier against movement of larger molecules. Each pore is surrounded by an elaborate proteinstructure called the nuclear pore complex, which selects molecules for entrance into the nucleus.

Entering the nucleus through the pores are the nucleotide building blocks of DNA and RNA, aswell as adenosine triphosphate, which provides the energy for synthesizing genetic material.Histones and other large proteins must also pass through the pores. These molecules have specialamino acid sequences on their surface that signal admittance by the nuclear pore complexes. Thecomplexes also regulate the export from the nucleus of RNA and subunits of ribosomes. DNA inprokaryotes is also organized in loops and is bound to small proteins resembling histones, butthese structures are not enclosed by a nuclear membrane.


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THICKNESS OF MEMBRANE = 60 - 90

THICKNESS OF PERI NUCLEAR SPACE = 100 - 1,000

In a growing or differentiating cell, the nucleus is metabolically active, replicating DNA and synthesizingrRNA, tRNA, and mRNA. Within the nucleus mRNA binds to specific proteins, forming ribonucleoproteinparticles. Most of the cells ribosomal RNA is synthesized in the nucleolus, a subcompartment of thenucleus that is not bounded by a phospholipid membrane (Figure 5-25). Some ribosomal proteins are addedto ribosomal RNAs within the nucleolus as well. The finished or partly finished ribosomal subunits, as wellas tRNAs and mRNA-containing particles, pass through a nuclear pore into the cytosol for use in proteinsynthesis (Chapter 4). In mature erythrocytes from nonmammalian vertebrates and other types of restingcells, the nucleus is inactive or dormant and minimal synthesis of DNA and RNA takes place. How nuclearDNA is packaged into chromosomes is described in Chapter 10. In a nucleus that is not dividing, thechromosomes are dispersed and not dense enough to be observed in the light microscope. Only during celldivision are individual chromosomes visible by light microscopy. In the electron microscope, thenonnucleolar regions of the nucleus, called the nucleoplasm, can be seen to have dark- and lightstainingareas. The dark areas, which are often closely associated with the nuclear membrane, contain condensedconcentrated DNA, called heterochromatin (see Figure 5-25). Fibrous proteins called lamins form a two-dimensional network along the inner surface of the inner membrane, giving it shape and apparently bindingDNA to it. The breakdown of this network occurs early in cell division, as we detail in Chapter 21.


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Function of Nucleus membrane

The nuclear membrane surrounds the nucleus that is covered with poresand it controls nuclear traffic and the nuclear membrane holds the nucleustogether.

The nuclear membrane encloses the nucleus of the cell that controls thethings which enters and leaves the nucleus. So it is also called as thenuclear envelope. Any material entering into the nucleus through nuclearpore should have a signal called "nuclear localizer signal" (NLS). This

signal which brings the material inside the nucleus is called "importinsignal" and protein which transports is called "nuclear importin protein".Similar is the case for "exportin signal" and "nuclear exportin protein"which transports material out of the nucleus.

The nuclear membrane pores regulate the exchange of materials betweenthe nucleus and the cytoplasm. Inside the inner membrane the nuclearlamina forms a network of filaments which play an important role in mitosisand meiosis.

In some eukaryotes a closed mitosis takes place in which the

chromosome remains within the nuclear membrane. Here the membraneitself undergoes a division as like the two daughter cells divide.

It separates the nucleoplasm from the cytoplasm and the nuclearmembrane ensures that the inside of the nucleus is isolated from the cellscytoplasm which allows two different environments to be maintained.

During prophase in the mitotic cell division, the nuclear membrane disintegratesand releases the chromosomes. At the end of Meta phase the nuclear membraneis not present and releases the nuclear lamina.

This is how nuclear membrane plays an important role in maintaining the stabilityof central controlling unit of cell called "nucleus".


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Nuclear lamina

The inner membrane of the nuclear envelope lies next to a layer of thin filaments which

surrounds the nucleus except at the nuclear pores. These may also serve as stabilizingfilaments. This structure is called the "nuclear lamina". It has the following structural andfunctional characteristics.

Consists of "intermediate filaments", 30-100 nm thick.These intermediate filaments are polymers of lamin, ranging from 60-75 kDA-type lamins are inside, next to nucleoplasm; B-type lamins are near the nuclear

membrane (inner). They may bind to integral proteins inside that membrane.The lamins may be involved in the functional organization of the nucleus.

They may play a role in assembly and disassembly before and after mitosis. After theyare phosphorylated, this triggers the disassembly of the lamina and causes the nuclear

envelope to break up into vesicles. Dephosphorylation reverses this and allows thenucleus to reform

Nuclear Pore Complexes (NPCs)

The nuclear envelope is perforated with thousands of pores. Each is constructed from anumber (30 in yeast; probably around 50 in vertebrates) different proteins callednucleoporins. The entire assembly forms an aqueous channel connecting the cytosolwith the interior of the nucleus ("nucleoplasm"). When materials are to be transported


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through the pore, it opens up to form a channel some 25 nm wide large enough to getsuch large assemblies as ribosomal subunits through.

The above figure shows a view of the nuclear pore from the top. It contains 8 subunitsthat "clamp" over region of the inner and outer membrane where they join. Actually, they

form a ring of subunits 15-20 nm in diameter. Each subunit projects a spoke-like unit intothe center so that the pore looks like a wheel with 8 spokes from the top. Inside is acentral "plug". The next (left) figure shows a cross section of the pore with the clamp-likecomplex adjacent to the membranes. The projected spoke is directed towards the central"plug' or granule.

Transport through the nuclear pore complexes is active; that is, it requires

energy many different carrier molecules each specialized to transport a particular cargo docking molecules in the NPC (represented here as colored rods and disks).

Import into the nucleus

Proteins are synthesized in the cytosol and those needed by the nucleus must be importedinto it through the NPCs.

They include:

all the histones needed to make the nucleosomes all the ribosomal proteins needed for the assembly of ribosomes all the transcription factors (e.g., the steroid receptors) needed to turn genes on

(and off) all the splicing factors needed to process pre-mRNA into mature mRNA

molecules; that is, to cut out intron regions and splice the exon regions.

Probably all of these proteins has a characteristic sequence of amino acids called anuclear localization sequence (NLS) that target them for entry.

Export from the nucleus

Molecules and macromolecular assemblies exported from the nucleus include:

the ribosomal subunits containing both rRNA and proteins messenger RNA (mRNA) molecules (accompanied by proteins) transfer RNA (tRNA) molecules (also accompanied by proteins) transcription factors that are returned to the cytosol to await reuse


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Both the RNA and protein molecules contain a characteristic nuclear export sequence(NES) needed to ensure their association with the right carrier molecules to take them outto the cytosol.

How does the nuclear pore complex work to transport

material in and out of the nucleus?

The pore serves as a water filled channel and has an effective diameter of 10 nm.Therefore, transport in and out of the nucleus can occur in several ways.

Diffusion

This can be tested by adding different sized molecules to the cytosol and watching therate of transport of each group. For example, molecules of:

5,000 MW are freely diffusable17,000 MW-- take 2 min to establish equilibrium44,000 MW--take 30 min to establish equilibrium60,000 MW--cannot move in by diffusion

This concept is important because it means that mature ribosomes (with both

subunits joined) cannot reenter the nucleus. Therefore, protein synthesis

(translation of mRNA) must occur outside the nucleus.

Active Transport

This form of transport is assumed when molecules larger than the pore diameter (10 nm)get into the nucleus. Studies with gold markers show that the pore can actually dilate upto 26 nm when it gets the appropriate signal. To understand the process, we need toanswer the following questions.

"Nucleoplasm"

The term "nucleoplasm" is still used to describe the contents of the nucleus. However, theterm disguises the structural complexity and order that we have seen here exist within thenucleus.

Genetic Organization of the NucleusThe configuration and organization of the DNA molecule within the cell nucleus influences themechanisms utilized by eukaryotic organisms for DNA replication. The processes of DNAreplication and modification in turn influence the structure and function of cells. In addition,because DNA codes genetic information for the transmission of inherited traits, the mechanismsregulating its replication and modification play a fundamental role in determining the geneticcharacteristics of an organisms offspring.


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Cooper Board Review Series Cell Biology

SBC 312 Genome Organizatio II(1)

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