L1: Architecture of the nucleus 1

intro, DNA nucleosomes and chromatin

How much DNA contained in nucleus

2 metres

What is the inside the nucleus

• chromatin

• equal amounts of protein and DNA

  • → to be able to see it

  • attracts dye easily which is fab

this is why chromatin is called chromatin 'chrome=colour

How small is the nucelus (where DNA contained)

10 um

• great degree of compaction

  • hierachy of structure

How is it so compact?

• genomic DNA associates with strucutrual chromatin components

  • histone proteins

  • forms ordered strucutres: nucleosomes

Further compaction of nucleosomes

• form higher order strcutures

• form chromosomes

What is around chromatin?

• surrounded by double lipid bilayer membrane system

• forms nuclear membrane

  • supported by protein meshwork: nuclear envelope

What is the role of nuclear pores

• regulated transport of macromolecules in and out

Role of the nucleus

information centre of the eukaryotic cell

• synthesise mRNA for protteins

  • → must be exported into cytoplasm for directing protein synthesis

• replicate its entire strucutre accurately during each cell division cycle

Nucleotide building blocks

Forming the phosphodiester backbone

The different bases

• either derived from Purine or Pyrimidine

• with some modifications

How these Base pairings make the grooves of the helix

• the major and minor grooves are important in gene expression and epigenetics

DNA and its strucutre: B form DNA wehn forms

Right-handed helix

When?

• physiologyical pH and salt concentration

What is the B-form of DNA? (skeletal model)

1. outer sugar-phosphate backbone chains connected via hydrogen-bonded base-pairs

2. forms major and minor groove

3. anti-parallel strands

4. phosphodiester backbones face OUT

5. paired bases face in

6. 10-11 bases per helical turn

7. Base accessibility in binding

   • high in major groove

   • less so if at all in minor groove

(Role of grooves)Access to genetic info is via

1. base-pair specific surface of the DNA helix accessible in grooves

needed for sequesnce -specific binding proteins

(Role of grooves) Access to epigenetic information is via

1. base modifications accessible in grooves

Interaction of DNA with sequence-independent binding proteins (e.g histones) also achieve through

• interaction with the backbone

Types of forms of DNA

1. B-form→ right handed double helix

2. A-form→ right handed

   • more open

   • major/minor grooves not as distinct

   • seen more in RNA

3. Triple helix

   • when B strand has added things to it

4. Z DNA

   • left-handed

   • GCGC etc flipped so different direction

Right handed double helix vs Left handed double helix

• to do with moving perpendiuclar finger hand up either arm thing?

How can DNA topology be visualied

• electron microscope

Small circular genomes of eurkayotic DNA viruses can be seen in 2 configurations

1. relaxed

2. supercoiled

e.g SV40, polyoma

1. Relaxed

• forms open relaxed ring

2. Supercoiled circle

• Helix coiled around itself

• forms more compact strucutre (compared to open relaxed) 

2. Use of supercoiling in bacteria

• to fit DNA into small cell

incontrast with…

2. Use of supercoiling in eurkaotic circular DNA

• consequence of its association with histone proteins 

• to form nucleosomes

Supercoiled→ relaxed

by DNA Topoisomerase enzymes

• transient breaking and resealing of the DNA backbone

DNA modifications usually to which base

• cytosine

• but can also be guanine

How cytosine modified

1. meythlation into DNMTs

2. oxygenated into TET

3. coniuted oxygenation

4. becomes more and more bulky

What does this result in?

• Cytosine sticks out

• epigenetic effect

Guanosine modifications can form

• secondary strucutures:

  • stable tetrad strucutre

  • in test tube and in cells

  • form higher order strucutres (see image)

• RESULT: plastic DNA modifications

What Electromircopy has shown about nucelosomes

• regular arrays of nucleosomes 

• along the genomic DNA→ 1/100 mm in diameter

  • → constituting a chromatin fibre

beads on a string

1. 30 micrometres physiology conditions

2. 10micrometres regular pattern

   • DNA linker shown

   • nucleosome blobs

What do certain DNA endonucleases do to DNA

e.g Micrococcal Nuclease (only cuts linker DNA)

• release nucleosomes as discrete substructures

• containing:

  • fragments of genomic DNA 

  • strucutrual histone proteins

How can the DNA and histones be separated further?

• high salt treatment

or

• denaturation

Although the core is 146 bp, further digestion with DNAse 1 shows…

• regular periodic patern of 10-12bp fragments

  • i.e number of base pairs per full helical turn

What does this indicate

• the DNA is wrapped around the surface of the histone core octamer

  • it is around the outside surface of the histone

  • not surrounded by the histone

  • this is beecause we got regular strands from this digestion

Digest the DNA with histone attached and see wht DNA is available→ with further digestion, will be on surface if there

• we get regular stands which means that it is turned 2 ½ times around the histone

• so must be on the surface

The nucleosomal wrapping of DNA around the histone core leads to…

• the formation of one contrained supercoil per nucleosome

→ this is released when histones are removed

• wrapped around twice

HIgher resolution of nucleosome core particles→ derived from cystallography and X-ray diffraction

front and side view

The histone unfolded

• shows the components

Nucleosomes core particles (in all eukarotic organisms)

1. 146 bp of DNA

2. octameric histone core:

   • with two copies of each core histone

   • (H2A, H2B, H3, H4)

How do histone proteins form stable substrucutures

1. Heterotetramers of two molecules of each:

   • H3 and H4

2. Heterodimers of

• H2A and H2B

The histone protein folds the handshake motif (can do with hands)

image shows an H2A and H2B handshake motif

These heterotetramers then form the histone octamer, how

• one tetramer

• two dimers

Role of histone octamer in nucleosome

• forms the strucutrual core

strucutre of nucleosome has been resolved at atomic resolution by

X-ray diffraction of pure nucleosome crystals

• high-resolution strucuture (image)

Details of this strucuture

• central glocular domains of histones proteins are located on inside

• N and C terminal tails of histones extend out from the core strucuture

How are nucleosomes assembled from histone subcomplexes? Stepwide assembly

1. tetramer of (H3-H4)₂ contacts DNA

2. this is subsequently joined by two H2A-H2B dimers

conditions for when this reaction occur?

• purified components

• in vitro

• if the salt conditions are carefully controlled

What about in vivo?

mediated by:

1. histone chaperones

2. chromatin assembly factors

(more detailed Stepwise assembly)

1. Handshake motif od H2A and H2B (twise to make two dimers)

2. Handshake motif of H4 and H3 (twice to make two dimers)

3. H3-H4 dimers binds to make H3-H4 tetramer

4. This attracts the DNA and its force around tetramer (knuckeles and fingers are in contact)

   • molecular velcro

5. two H2A-H2B dimers bind

6. Histone octomer is made

7. NEXT adjacent nucleosomes connect with DNA linker

How are adjacent nucleosomes connected?

• by linker DNA

Size of linker DNA?

varies between:

1. cell types

2. tissues

3. species

Associations of linker histone H1 with the linker DNA and nucleosome core are involved in forming…

• higher order structures

Modifications of histones, how?

• tailes of the four core histones  extend out from the nucleosome core

• available to be acted upon

• by protein modifying enzymes

• Specific amino acids in the tails are often post-translationally modified by a wide variety of modifcations

Histones are proteins, and have N and C termini.

The N termini stick out a bit and so can be modified

e.g with acteyltransferase

What modifications can be done to thse amino acids?

1. acetylation

2. methylation

3. phoosphorylation

4. ubiquitination

Lysine acetylation and methylation

1. Acetylation→ neutralise charge

2. Methylation→ fixed, and bulky

Arginine acetylation and methylation

1. Acetylation→ lose charge and big

2. Methylation

Serine phosphorylation

What do these modifications do?

comprise epigenetic information

• heritable information that is ot encoded in the DNA sequence

  • genetic locus is activated/inactivated

  • under given set of cellular circumstances

What is this concept known as?

• histone code

What happens to this hitone code?

1. ‘read’ by proteins that bind specifically to modified histone tailed

e.g of this?

Lysine 9 of histone H3

• Acetylated:

  • Specified active state:

• Trimethylated:

  • Specified inactive state

by attracting different binding proteins

Permenet/reversible?

• Sates are reversible

• modifications cna be removed and new modification can be added

Therefore the cell’s chromatin is

• dynamic entity

• regulated changse in its strucutre occur as it

  • responds to their environment

  • grow

  • differentiate