1. DNA Packaging

2. Objectives DNA packaging in prokaryotes.
How and why eukaryotic DNA is packaged.
Super coiling is essential for DNA packaging.
The role of histone in packaging.
How DNA packaging is adjusted during replication and transcription.

4. Prokaryotes small size of genome
circular molecule of naked DNA called a PLASMID
DNA is readily available to RNA polymerase
control of transcription by regulatory proteins (operon)
most of DNA codes for protein or RNA
no introns, small amount of non-coding DNA
regulatory sequences: promoters, operators
Plasmid

5. DNA packaging in Prokaryotes

6. Eukaryotes much greater size of genome located in nucleus
how does all that DNA fit into nucleus?
DNA packaged into chromatin fibers
regulates access to DNA by RNA polymerase
most of DNA does not code for protein
97% “junk DNA” in humans

8. DNA Packing DNA coiling & folding Double helix Nucleosomes
How do you fit all that DNA into nucleus of a eukaryotic cell?
DNA coiling & folding
Double helix
Nucleosomes
Chromatin fiber
Looped domains
Chromosome
from DNA double helix to condensed chromosome

9. Organization of Eukaryotic DNA
Genes that store the cell's information and instructions are made of DNA sequences
In eukaryotic cells, DNA is packaged with proteins to form chromatin fibers that make up chromosomes
This organization of eukaryotic DNA allows DNA to be accurately replicated and sorted into daughter cells without much error and tangling during cell division

10. Eukaryotic DNA is associated with tightly bound proteins  Histones

11. Histones and the formation of nucleosomes
Five classes of histones, designated H1, H2A, H2B, H3, and H4
These small proteins are positively charged at physiologic pH as a result of their high content of lysine and arginine
Because of their positive charge, they form ionic bonds with negatively charged DNA
Histones, along with positively charged ions such as Mg2+, help neutralize the negatively charged DNA phosphate groups

12. - Organization of Human DNA -

13. Nucleosomes “Beads on a string” 1st level of DNA packing
histone proteins
8 protein molecules
many positively charged amino acids
arginine & lysine
DNA backbone has a negative charge
histones bind to DNA due to a positive charge

14. Nucleosomes  Basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores
Two molecules each of H2A, H2B, H3, and H4 form the structural core of the individual nucleosome “beads”
Around this core, a segment of the DNA double helix is wound nearly twice, forming a negatively supertwisted helix

15. Neighboring nucleosomes are joined by “linker” DNA approximately 50 base pairs long
Histone H1, of which there are several related species, is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads
H1 is the most tissue-specific and species-specific of the histones
It facilitates the packing of nucleosomes into the more compact structures

17. 30 nm fibre (Solenoid Fibre)
Nucleosomes are organized in a stacked spiral structure
The solenoid fibre is known as the 30 nm fibre

18. Higher levels of organization
  Nucleosomes can be packed more tightly to form a polynucleosome (also called a nucleofilament)
This structure assumes the shape of a coil, often referred to as a 30-nm fiber
The fiber is organized into loops
Additional levels of organization lead to the final chromosomal structure

20. DNA Supercoiling

21. Chromatin Packing Euchromatin Heterochromatin eu – true
loosely packed DNA regions which allows transcription to readily occur
hetero – different
tightly packed DNA regions with little transcription

22. DNA packing and transcription
Degree of packing of DNA regulates transcription
tightly packed = no transcription
= genes turned off
darker DNA (Heterochromatin) = tightly packed
lighter DNA (Euchromatin) = loosely packed

23. Cellular DNA must be very tightly compacted just to fit into the cell
This implies a high degree of structural organization
It is not enough just to fold the DNA into a small space, however
The packaging must permit access to the information in the DNA for processes such as replication and transcription

24. The term "super coiling" means literally the coiling of a coil
The term "super coiling" means literally the coiling of a coil. A telephone cord for example, is typically a coiled wire

25. DNA is coiled in the form of a double helix
A bending or twisting of that axis upon itself is referred to as DNA supercoiling
DNA supercoiling is generally a manifestation of structural strain
Conversely, if there is no net bending of the DNA axis upon itself, the DNA is said to be in a relaxed state

26. Replication and transcription both require a transient separation of the strands of DNA, and this is not a simple process in a DNA structure in which the two strands are helically interwound

27. DNA is referred to as Negatively supercoiled  when it is underwound
Positively supercoiled  when it is overwound
Negative supercoiling plays a key role in allowing the
DNA of the chromosomes to be compacted  fit inside the confines of a microscopic cell nucleus  because negative supercoiled DNA is underwound , it exerts a force that helps separate the two strands of the helix  which is required by both replication ( DNA synthesis) and transcription ( RNA synthesis)

29. Cells rely on enzymes to change the supercoiled state of a DNA duplex
These enzymes are called Topoisomerases  because they change the topology of the DNA
Cells contain a variety of topoisomerases, which can be divided into two classes
Type 1 Topoisomerases  change the supercoiled state of a DNA molecule by creating a transient break in one strand of the duplex
The enzyme cleaves one strand of the DNA and then allows the intact, complementary strand to undergo a controlled rotation, which relaxes the supercoiled molecule

30. Topoisomerase I  essential for processes such as DNA replication and transcription  it functions in these activities by preventing excessive supercoiling from building up as the complementary strands of a DNA duplex separate and unwind

32. Type II topoisomerases  make a transient break in both strands of a DNA duplex
Another segment of the DNA molecule or a separate molecule entirely is then transported through the break, and the severed strands are released