Chromosomal Packaging

Fatih EREN
Medical Biology Department
fatiheren@marmara.edu.tr
Definitions

• Genome, Gene and Chromosome.

• These three terms are different from each other.

• The common feature of them is to be made up of the most

important molecule to life on earth: DNA
Genome (Gene + Chromosome)

• Genome is the whole genetic material of the organism. More than all the genes.

• Genome usually consist of DNA.

• However, in RNA viruses, Genome consist of RNA.

• Genome term was coined by German Botanist Hans Winkler in 1920.

• He derived the term simply by combining gene and final syllable of chromosome.
 Genome
• Hans Winkler has identifed the genome as all genetic material in the cell of organism.

• Eukaryotic genome consist of nuclear DNA, mitochondrial DNA and chloroplast DNA localized in the nucleus,
mitochondrion and chloroplast, respectively.

• In prokaryotes, genetic material is localized in the space called nucleoid. As you know, prokaryotic organisms
contain no membrane-bound organelles. Therefore, the nucleoid has no membrane around it.

• Some bacteria have auxiliary genetic material. They are called plasmid.

• Therefore, plasmid DNA is also part of prokaryotic genome.

Genome
• The nucleoid is mostly composed of multiple compacted copies of DNA in a
continuous thread, with the addition of some RNA and proteins.

• The DNA in prokaryotes is usually double-stranded and generally takes a circular

shape.

• Bacterial genomes have nucleoid associated proteins that are thought to help package
the genome into a nucleoid,

• but the precise functions of these DNA-associated proteins are not understood.
 Does genome size represent the development of an organism ?

• You can see the genome size of organisms from viruses to eukaryotes.

• Genome size is quitely variable between organisms.

• Viruses and bacteria tend to have very small genomes

• Prokaryotes typically have smaller genomes than eukaryotes

• Sizes of plant genomes can vary dramatically due to the capacity for
plant species to self-fertilise and become polyploid

Variation in Genome Sizes For Different Types of Organisms

 Does genome size represent the development of an organism ?

• When you examine the graph well, you will realize that many other
eukaryotes have larger genomes than humans.

• Although the human genome is 3.5 billion base pairs, the canopy plant Paris
japonica has a genome of 150 billion base pairs.

• In the light of such information, we can say that genome size associated with
complexity of organism. However, Genome size is not always related with
genetic complexity in the eukaryotes Variation in Genome Sizes For Different Types of Organisms
 Genome and Cell Size

• Generally organisms genome too long to fit into the cell

• For example: Escherichia coli cell is 1-2 µm long, and about 0.5 µm diameter.
• We can calculate whether E.Coli genomes fit into the E.Coli cell.
• We know that Each base pair is around 0,34 nanometers long and E.Coli genome size is 4.6 million bp.
• According to these informations, the length of E.coli DNA is [(0,34 nm x (4,6 x 106)] about 1,6 mm.
• Although E.coli is a prokaryote, its genome size is about 1000 times bigger than its cell size.
 Genome and Cell Size

• When it comes to cells size, we encounters the interesting findings.

• Generally, organisms’ genome too long to fit into the cell.
• This is important problem that needs to be resolved.
• Moreover, you can see easily that in this illustration, The size of the eukaryotic cell nucleus is almost the
same as the cell size of the prokaryotic organism.
 Viral Genomes
• Viral genomes can be consisted of either DNA or RNA .

• The genomes of DNA viruses can be either single or double

stranded DNA.

• Most DNA virus genomes is made of single stranded and

linear molecule of DNA, but some are composed of a circular
DNA molecule.

• In viruses, there is not nucleus, but viral genome is packed

inside capsid or core proteins.

• The viral genome and its capsid proteins together are called
nucleocapsid
 Viral Genomes
• For example: Hepatitis B Virus genome is partly circular double stranded DNA of about 3.200 nucleotide pairs.

• HBV infection is the reason of acute and chronic hepatitis.

• HBV infection can also lead to serious liver disease complications such as cirhosis and hepatocellular carcinoma.

• Currently HBV infection is still important health problem for our country and world.

• RNA viruses can have either single or double stranded RNA genome.

• For example: SARS-CoV-2 genome is single stranded RNA of about 29.900 nucleotide.

• Specific genetic sequences (E,N and S gene) within the SARS-CoV-2 genome are used to detect the COVİD-19 specifically.
Chromosome
• Organism's genome must be packaged to fit into cell because of their genome size.

• Basic organisations of chromosomal packaging are loop and coil formations.

• Moreover, genome must be protected during the cell division.

• DNA is a working molecule; it must be replicated when a cell is ready to divide,

• and it must be “read” to produce the molecules, such as proteins, to carry out the
functions of the cell.
 What are the advantages of Chromosome structure

Specific proteins are Chromosomes help a cell

needed to package of Therefore chromosomes to keep a large amount of
genome, and some of consist of genetic material genetic information,
them also participate the and specific proteins. functionally organized, and
formation of chromosome. compact
 The chromosome • In a few very simple organisms such as bacteria, the entire genome is
usually packaged into the single chromosome.
number of organism is
not also always • In organisms with larger genome than bacteria, genome packaged into
related with the multiple different chromosomes.

complexity of organism • Closely related species tend to have a similar number of chromosomes.

directly. • For example, chimpanzees, our closest species have 48 chromosomes in

each of their cells.

• Higher organisms do not always have more chromosomes.

• For example, some species of ferns have over 1200 chromosomes while
goldfish has 94 chromosomes.
 Prokaryotic Chromosome
• Prokaryotic Genome is double stranded DNA.

• Prokaryotic genome is usually single and covalently closed circular. Moreover,

there are extra circles called plasmids.

• Prokaryotic genome is usually packaged into single, circular chromosome.

• However, some bacteria such as agrobacterium tumofaciens, have multiple

cromosomes (one linear and one circular chromosome).

• Moreover, many bacteria such as borrelia, have linear chromosomes and linear
plasmids.

• Prokaryotic DNA is supercoiled and compacted by enyzmes and nucleoid

associated proteins.
Bacterial Chromosomal Packaging

• In Prokaryotic cells,
• various nucleoid associated proteins (NAPs)
• nucleoid associated RNAs naRNAs
• and specific enzymes ( DNA topoisomerases)
• are responsible for producing a complex, compacted bacterial chromosome.

• In Bacterial chromosomal packaging occurs in the certain hierarchical organization.

• DNA supercoiling
• Interaction of DNA with nucleoid-associated proteins
• Structural maintenance of chromosomes (SMC) complexes
 How to discriminate the positive and negative supercoils ?

Supercoilings provide the condensation of cccDNA therefore we should know the types of supercoilings.
At the end of this process extra coiling occurs and therby cccDNA shortens and become compact.
This coilings are also called writhe
1. First, we divide the circular relaxed DNA into two regions with an axis.
2. To form the negative supercoiling, First, the DNA duplex to the right of the axis is coiled over the other DNA duplex located to the left of the axis.
3. In resulting structure, overlying duplexes are always clockwise.
4. To form positive supercoiling, all the steps above occur in the opposite direction.
 How to discriminate the positive and negative supercoils ?

1. First, we divide the circular relaxed DNA into two regions with an axis..
2. To form the positive supercoiling, the DNA duplex to the left of the axis is coiled over the other DNA
duplex located to the right of the axis.
3. In resulting structure, overlying duplexes are always counterclockwise.
Topological Linking Number (Lk): Twist (Tw) + Writhe (Wr)
Twist
• Tw = “the number of times one strand of DNA duplex crosses another strand of it. 2
3

• Twist means that the number of full helical turns of DNA strands.

• Wr = “the number of times DNA duplex crosses another duplex” 4

• Writhe means that the number of full helical turns of DNA duplex or
supercoiling. 1
• There are six Twist but not writhe.
• Lk= 6+0→Lk=6
• Linking number in the relaxed circular DNA is called basal linking number and is 5
indicated with Lk0 symbol. 6
 Topological Linking Number (Lk): Twist (Tw) + Writhe (Wr)
• Writhes occurs during the supercoiling process.
• Writhe (Wr) is coiling of the DNA double helix on its axis that requires bending.
• Writhes can be resulting from negative supercoling and it is numbered minus (-).

• Writhes can also be resulting from positive supercoling and it is numbered positive (+).
• In the relaxed cccDNA, Lk0:36+0=36.
• In the negatively supercoiled DNA, Lk=36 Lk:36+(-4)=32
• ΔLk: Lk-Lk0: 32-36=-4
• A decreasement in the linking number (Lk<Lk0) creates negative supercoiling
• Whereas an increasement in the linking number (Lk>Lk0) creates positive supercoiling
How to produce supercoiling in the process bacterial choromosomal packaging.

DNA topoisomerase produces negative supercoiling to compact the bacterial DNA

Topological Linking Number (Lk): Twist (Tw) + Writhe (Wr)

• Writhes can adopt two structures; plectoneme and solenoid or toroid.

• A plectonemic structure arises from the interwinding of the helical axis.
• Bacterial DNA is usually negatively supercoiled and folded into plectonemic writhes (loop).
DNA Supercoiling responsible for
chromosomal condensation and organization

• Plectonemic supercoils represent effective supercoiling of the bacterial genome

• Supercoiling also helps bring two distant sites of DNA in proximity thereby promoting a potential functional interaction
between different segments of DNA.
• Other topoisomerases including topoisomerase I, III, IV, removes negative supercoiling.
• Topo I is responsible for relaxation of 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 bacteria.
• Both enzymes are essential for bacteria survival.
Interaction of • Although, the chromosomal DNA is on average negatively supercoiled and folded
into plectonemic loops,
DNA with
nucleoid- • The size of bacterial choromosome is still not being compacted to fit into nucleoid
associated which is less than a micron.
proteins
• Moreover, Chromosomal DNA must not be only condensed but also functionally
(NAPs) organized in a way that is compatible with DNA transaction processes such
as replication, recombination, segregation, and transcription.

• There are need additional factors for packaging chromosomal DNA.

Interaction of DNA with nucleoid-associated
proteins (NAPs)
• Bacteria do not have histone proteins like being in the eukaryotes.
• However, in the nucleoid, NAPs are highly abundant and constitute a significant proportion of the protein.
• NAPs are
• HU: Histone like proteins
• IHF: Integrating Host Factor
• H-NS: Heat-stable Nucleoid Structuring protein
• Fis: Factor for Inversion Stimulation.
• A double-stranded DNA tends to remain at maximum length despite the bending forces (such as
supercoiling). The maximum length is called persistence length.
• NAPs induce and stabilize bends in DNA, thus aid in DNA condensation by reducing the persistence length.
• NAPs condense and organise DNA by bending, bridging, wrapping, and bunching.
 Interaction of DNA with nucleoid-associated proteins(NAPs)

• NAPs bind to the chromosomal DNA mostly in the non-sequence specific mode and it is this mode that is crucial for chromosome compaction
• Moreover, NAPs participate in chromosome compaction is by constraining negative supercoils in DNA thus contributing to the topological organization
of the chromosome.
• The plectonemic loops combine into six spatially organized domains (macrodomains).
• Macrodomains are defined by more frequent physical interactions among DNA sites within the same macrodomain.
• Long- and short-range DNA-DNA connections formed within and between the macrodomains contribute to condensation and functional
organization.
• Finally, the nucleoid is a helical ellipsoid with regions of highly condensed DNA at the longitudinal axis.
 Structural Maintenance of Chromosome proteins
(SMCs) turns supercoiling into loops.
• SMC function as a dynamic nucleoid scaffold in bacteria (also in
chromosome segregation)
• They belong to a family of ATPases,
• Two MukB monomers that are smc proteins, associate via continuous
antiparallel coiled-coil interaction
• This interaction forms 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 that are MukE and MukF, join with MukB.
• 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), like daisy flower.
 Gene

• Gene is the part of the genome.

• Gene is the coding sequences of the DNA molecule.
• In other words, Gene is spesific DNA sequence within Genome.
• Gene encodes functional products such as non-coding RNA and proteins
• Most genes are both transcribed and translated. These genes encode all
proteins.
• The other genes can be only transcribed such as transfer RNA and ribosomal
RNA genes so on. They encodes non-coding RNA (ncRNA).
 Is there a correlation between number of the
genes and morphological complexity ?

The number of genes increase from prokaryotic organisms to eukartyotics.

However, gene number is unrelated the complexity of the organisms.

Prokaryotic Genome &
Eukaryotic Genome
• Prokaryotic Genome
• Most Genome is consisted of protein coding sequences
• Non-coding sequence is less than %10 of genome.
• Eukaryotic Genome
• Protein coding sequence is only about %2 amount of
genome.
• The other remaining part of genome (%98) is consisted
of non coding and repetitive DNA sequences including
regulatory sequence, introns and so on.
 • Eukaryotic genome is double stranded DNA.

• Eukaryotic genome is diploid. Eukaryotes cells contain two sets of

chromosomes that are inherited from parents.
What are the
• Eukaryotic Genome is located in the nucleus that is surrounded by
features of nuclear membrane.

Eukaryotic • Eukaryotic genome is usually multiple and linear. Moreover there are
Genome? extra organelle’s genome such as mitochondrial and chloroplast
genomes.

• Eukaryotic genome is usually packaged into multiple , linear

chromosome.

• Eukaryotic genome is compacted by histone proteins and non-histone

proteins.
What is the average length of DNA
molecule in a human cell ?
• The haploid genome (23 chromosomes) contains
approximately 3.4 billion base pairs of DNA.

• Therefore, there is a total of 6 billion base pairs of

DNA per diploid somatic cell.

• Each base pair is around 0,34 nanometers long

(a nanometer is one billionth of meter)

Then, the length of DNA is

[(0,34x10-9 x (6 x 109)] about 2 meter.
How is human DNA fit into a tiny nucleus?

• As you know, each cell nucleus contains 2

meters of DNA.
• However, cell nucleus,where DNA is located, is
10-20µm in diameter.
• Therefore, nuclear DNA must be packaged to fit
into nucleus.
• In Eukaryotes, chromosomal packaging
occurs in the certain hierarchical organization.
Main technics of chromosomal packaging

The essential component is nucleosome bead and basic repeating unit of

chromatin.

Nucleosome beads serve as the spool

around which the DNA is wrapped

Nucleosome beads are

made of histone proteins
What are the
Special Proteins for Packaging
of Human Nuclear DNA ?
 • Each histone is small proteins.

• They contains only about 100 amino acids.

• The total amount of histone in chromatin roughly

equals the mass of DNA

HİSTONES • They are positively charged proteins

• DNA is negatively charged, due to the phosphate groups

in its phosphate-sugar backbone, so histones bind with
DNA very tightly.
The importance of HİSTONES

• Strikingly, Histones structure are very similar among eukaryotes; for

example, cow H4 is almost identical to pea H4. There are just two amino
acid of difference between them.

• The apparent conservation of histone genes during the evolution probably

reflects the important role of histones in organizing DNA within cells.
Classes of Histones

• There are two main classes of histones:

• Core Histones

• Linker Histones
 11 nm

• Each nucleosome bead two copies of H2A, H2B ,H3 and H4.
• They form a histone octamer that is also called the
nucleosome core.
• Nucleosome bind and wraps (pack) about 146 base pairs of
DNA around itself.
• Nucleosome bead and DNA is the basic form of chromatin.
• DNA is thus packaged in a lineer form.
Nuclesomes
(First Level Packaging)
• Nucleosome bead is the basic repeating unit of chromatin.

• It provides the lowest level of compaction of double-

stranded DNA into the cell nucleus.

• In an electron microscope, isolated chromatin frequently

looks like “beads on a string”.
Linker Histones
DNA Exit
Linker histones:
• H1
• H5 DNA
Enterance
• H1 is called the linker histone.
• H1 binds to DNA (an average of about 20 base pairs) that is
located at the enters and exits of each nucleosome core.
• Linker histones performs the function as a clamp.
• Linker histone is being necessary for the condensation of
nucleosome chains into higher order structures,
• H5 performs the same function as H1 and replaces H1 in
certain cells.
 • The coiling of 146 base pairs is not very long for condensantion, considering that each
chromosome contains over 100 million base pairs of DNA on average.

Chromatosome • The addition of H1 protein and Linker DNA to nucleosome form a chromatosome.

• This binding of H1 protein to linker DNA wraps another 20 base pairs around nucleosome
beads.
Second Step: 30 nm Chromatin
Fiber
• The packaging of DNA into nucleosomes shortens the
naked DNA length about sevenfold.

• In other words, haploid DNA that is 1 meter long will

become a chromatin fiber just 14 centimeters long.

• Despite this shortening, chromatin fiber is still too long to

fit into the nucleus, which is typically only 10 to 20 microns in
diameter.

• Therefore, there is need to further coil the chromatin fiber.

30 nm FIBER

•Chromatin can be further compacted into higher order structures by forming solenoidal
coil

• Six chromatosomes are joined by H1 to form solenoidal coil. The interactions between
the histone tails of each of chromatosome contribute the formation of solenoidal coil. 30
nm DNA fibril occurs at the end of this process.

•30-nm fiber is quite prevalent in the interphase nucleus.

•At this level, Naked DNA is compacted 40 times.

Third step: 300 nm FIBER
•The 30nm fibers forms loops anchored to
a nonhistone protein scaffold.
• The looped structures form the interphase
chromosomes.
• Each loop contains 50 solenoid coils.
• Occuring chromosome is 300-nm
diameter.
•The scaffold is rich in topoisomerase II
(non histone protein), and H1 molecules
also appear to be present.
 Metaphase Chromosome
In a mitotic chromosome, the looped domains
themselves coil and fold in a manner not yet
fully understood,

Further condensing of loop structure forms

minibands.

Each minibands contains 18 loop. Each loop

containing over a million base pairs.

The DNA in these minibands has been

compacted by about 10,000-fold!
Minibands
Further compacting all the chromatin to
produce the characteristic metaphase
chromosome (also shown in the micrograph.
The width of one chromatid is 700 nm.

Particular genes always end up located at the

same places in metaphase chromosomes,

indicating that the packing steps are highly

specific and precise
 The structural organization of
the core histones

•N terminal tails of each histone molecules protrude from the nucleosome and are accessible to enzymes that add and
remove chemical groups such as methyl and acetyl groups.
•The addition of these group can change the level of expression of genes packaged in nucleosome.
The effects of histones on
replication and transcription

In the cell cycle, the histones leave the DNA only briefly
during DNA replication.

Generally, they do the same during transcription, This

process is required for binding of transcription factor to
promoter region of DNA.

Nucleosomes, and in particular their histone tails, play an

important role in the regulation of gene expression.
 Chromatin Remodelling
for Transcription

• The main chromatin remodelling mechanism is acetylation

of lysine residues at the N terminal of histone proteins.

• Acetylation, mediated by histone acetyltransferases (HATs),

eliminates the positive charge on the lysine and thereby
decreases the interaction of the histone with the negatively
charged DNA.

• Removal of the acetyl group by histone deacetylases

(HDACs) restores the positive charge on the aminoacid and
fosters stronger interactions between histones and DNA.
 They serve as structural roles
NON-HISTONE
CHROMOSOMAL
PROTEINs They play role in genetic
processes such as transcription
and replication.
Scaffold Proteins

• Scaffold proteins provide or support with a raised

framework or platform.

• A protein whose main function is to bring other

proteins together for them to interact.
 Experiments revealed the Scaffold Proteins

When the chromosome is treated with a

concentrated salt solution.

This treatment removes histones and most of

the other chromosomal proteins

There are only remained a “skeletons/scaffold

proteins to which DNA was attached.

These scaffold proteins play a role in the

folding and packaging of chromosome.