Molecular BiologyGenome & GenomicsChromatin Structure

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190018. India.

• Prokaryotes have a simple genome in their cells

• The chromatin of an E. coli is a single double-stranded circular DNA molecule, lying usually in the centre of the cell without any surrounding membrane

• This region of the cell known as nucleoid –characterized by a very high density of DNA

• It is made up of ~4.5 million bps and remains associated with the proteins

• The DNA consists of 50-100 domains or loops each of about 50-100bp in size.

• The overall E. coli chromatin was found to be highly negatively supercoiled

• Also small basic dimer protein called as HU protein is associated with the DNA domains to further constrain it. This protein is analogous to eukaryotic histone proteins

E. coli

E. coli

Eukaryotic Chromatin

• The structure of eukaryotic genome is much more complex than that of prokaryotes and remains well within the distinct structure called as nucleus

• In eukaryotes genome is divided into chromatin threads which on condensation forms a distinct double armed entity called as chromosome seen usually during the metaphase stage of mitosis

• The chromosome contains two arms called as sister chromatids joined at the point called as centromere. The tips of each chromatid are called as telomeres which are actually the ends of DNA molecules

• Centromere is the site of attachment of spindle fibres to kinetochore-which is assembled & located within it. In yeast centromeric DNA is a tandem repeat (over thousand copies in same orientation) of a short AT-rich sequence of 88bp

• Telomeres are the specialized DNA sequences that form the ends of the eukaryotic chromosomes. They help to stabilize the chromosomes by preventing the ends from degrading effects during division

• Telomeres are synthesized by enzyme called as telomerase. A telomere consists of upto hundreds of copies of short repeated sequence of 5’-TTAAGGGG-3׳ (humans) 5׳-TxGy-3’ (yeasts, x & y = 1-4)

Eukaryotic Chromatin

• The chromosome structure is characterized by three parameters, each of which is defined by two values - p and q• p represents short arm of chromosome • q represent the long arm

• d-value: It is the difference between the lengths of the two arms of the chromosome

d-value = q - p• r-value: It is the ratio of the length of the long

arm to that of the short arm of the chromosome

r-value = q / p• i-value: Referred as the centromeric index,

represents the distance from the centromere to the tip of the short arm. it is expressed as the percentage of the total chromosomal length

i-value = 100p / (p+q)

Eukaryotic Chromatin

• During the functional state of the cell which is called as theinterphase, cells genome is not present in the condensed chromosome form but exist largely as the unwound thread like structure called as chromatin thread

• It remains associated with proteins categorized into two types non-histones & histones

• The non-histone proteins include enzymes involved in DNA and histone metabolism, replication, recombination and transcriptional regulation.

• They also include the scaffold proteins which organize higher order chromatin structure, and the high mobility group proteins (HMG proteins)

• A further class of non-histone proteins, termed prota­mines,facilitates the packaging of DNA into the sperm head

DNA in Functional State of cell

• Most of the protein in eukaryotic chromatin consists of histones, of which there are five families, or classes: H2A, H2B, H3 and H4, known as the core and H1

• The core histones are small proteins, with masses between 10 & 20 kDa

• All histone proteins have a large positive charge; between 20 and 30% of their sequences the basic amino acids, lysine and arginine

• Because of the presence of highly basic residues histones interact with the DNA by forming salt bridges with negatively charged DNA backbone

Histones

• All Histones possess an inherent ability to associate & form various complexes in absence of DNA, notably (H3-H4)2 tetramer and the H2A-H2B dimer.

• Each histone contains a three α helix motif called a histone fold which facilitates these interactions besides there is also an N-terminal tail protruding from each histone.

Histones

• H1 histones are somewhat distinct from the other histone classes in a number of ways

• Are a little larger at around 23 kDa

• In addition, there is more variation in H1 sequences both between and within species than in the other classes

• Are more easily extracted from bulk chromatin,

• Are present in roughly half the quantity of the other classes, of which there are very similar amounts

• Is not associated with the core assembly

Histones

• Treatment of chromatin with micrococcal nuclease, an endonuclease which cleaves double-stranded DNA, led to the isolation of DNA fragments with discrete sizes, in multiples of approximately 200bp, called as mononucleosomes

• It was discovered that each 200 bp fragment is associated with an octamer core of histone proteins, {(H2A)(H2B)(H3)(H4)}2 called as the core histones, and more loosely with one molecule of H1

• Prolonged digestion with nuclease leads to the loss of H1 and yields a very resistant structure consisting of 146 bp of DNA associated very tightly with the histone octamer

• This structure is known as the nucleosome core, and is structurally very similar whatever the source of the chromatin

Nucleosome

Nucleosome

• The histone octamer forms a wedge-shaped disk

• Around this 146 bp of DNA is wrapped in 1.8 turns in a left-handed direction

• One molecule of histone H1 binds to the nucleosome, and acts to stabilize the point at which the DNA enters and leaves the nucleosome core

• In the presence of H1, a further 20 bp of DNA is protected from nuclease digestion, making 166 bp in all, corresponding to two full turns around the histone octamer

• A nucleosome core plus H1 is known as a chromatosome

• The core particles are joined together by linker DNA, whose length varies between species & between various cells

Nucleosome

Nucleosome

Nucleosome

• Chromatin present in the interphasic nucleus is of two main forms:

• 1. Euchromatin- which is thought to comprise only 30nm fibers and is usually present in the nucleoplasm of the nucleus.

It is the region of active genes that is the genes that are functional and are actively transcribing

• 2. Heterochromatin- which is a highly condensed chromatin of high order structure and tends to cluster around the nuclear periphery

It is usually transcriptionally repressed, and may be constitutive (condensed at all times and found near centromere) or facultative (repressed in some cells)

Chromatin Compaction

• Depending on the manner in which single stranded complementary DNA (which is previously melted) re-associates in the mixture DNA of eukaryotes is divided into three categories reflective of the three phases of the cot curve:

• Unique sequence DNA: This fraction of genomic DNA represents the slowest phase of the Cot curve.

• It represents the coding regions of the genome of the organism which occur in only one or a few copies per haploid genome.

Genome Complexity

• Moderately repetitive DNA: This fraction of the genome represents the middle phase of the cot curve & consists of a number of types of repeated sequence.

• This fraction actually represents those genes whose products are required in unusually large quantities by the organism. One example is the RNA-encoding genes (rDNA).

• The gene which encodes the 45S precursor of the 18S, 5.8S and 28S rRNAs, for example, is repeated in arrays containing from around 10 to around 10000 copies depending on the species.

• In humans, the 45S gene occurs in arrays on five separate chromosomes, each containing around 40 copies.

Genome Complexity

• Highly repetitive DNA: It represents the first phase of the cot curve which reanneals very fast during the reaasociationanalysis; in eukaryotic genomes

• This fraction consists of very short sequences from 2 bp to 20-30 bp in length, in tandem arrays of many thousands of copies.

• Such tandem repeats are called as satellite DNA, they are named so because they were identified as distinct bands with buoyant density different from that of the bulk DNA in density gradient centrifugation.

• The predominance of highly repetitive DNA varies between species, but typically represents 10-30% of the genome

• Because of its low complexity, it is sometimes termed simple sequence DNA

Genome Complexity

Questions?

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