DNA Packaging

1
Chapter 16

• DNA Packaging

2
Prokaryotic Packaging

• Bacterial chromosomes and plasmids
• Typically circular in the nucleoid no membrane
  but a distinct area
• DNA is negatively supercoiled in a variety of
  loops can alter one loop without hurting other
  looped supercoils
• RNA and proteins thought to hold together
• Nuclease treatment releases a loop but not the
  supercoil
• Topoisomerase (catalyze interconversion between
  relaxed and supercoil) relaxes supercoil
• Loops of DNA association with basic protein
  similar to histone

3
Bacterial DNA
4
Plasmids

• Small circular DNA encodes genes for own
  replication and 1 or more cellular functions
• 3 classes of plasmids
• F fertility bacterial conjugation
• R resistance drug resistance
• col colicinogenic secrete colicins that kill
  bacteria that are col negative
• Occasionally E coli has cryptic plasmids with
  unknown function

5
Eukaryotic Packaging

• DNA in chromatin or chromosomes
• Have more than 1 DNA, 1 may be 10 cm long
• DNA protein chromatin
• During cell division, compact into chromosomes
• Structural complexity greater amounts of
  protein
• Protein is histone that contain high amounts of
  lysine and arginine
• Positively charged AA to bind negative DNA -
  ionic bonds
• Mass of protein is mass of DNA

6
Histone Proteins

• 5 types of histone proteins
• H1 ½ of other histone proteins
• H2A
• H2B
• H3
• H4
• Concentration in most eukaryotic cells
• Also have non-histone proteins have enzymatic,
  structural and regulatory roles

7
Nucleosomes

• Have a repeating structural subunits seen in DNA
  or histones
• Have a bead (histone) on a string (DNA) appearance

8
Proof of Nucleosome

• Conclusions
• Chromatin proteins clustered along DNA in a
  regular pattern - 200 bp
• DNA located between these protein clusters is
  susceptible to nuclease digestion

9
Additional Findings

• Using a different nuclease, centrifugation and EM
  to see various clusters of particles DNA was
  200 bp long and larger ones were 400 bp and so
  forth
• 200 bp and protein is the nucleosomes

10
Nucleosome Core

• Made up of the histone octamer
• H2A and H2B are a unit and H3 and H4 are a unit
• Contains 8 histone proteins, 2 of each one
• Core octamer has 146 bp of DNA (1.7 x around
  core), remaining 64 bp are linker DNA between the
  nucleosomes
• This is were H1 interacts, associated with the
  linker DNA

11
Chromatin Fibers/Chromosomes

• Nucleosome formation is first step in DNA
  packaging
• Ususally a 30 nm thick strand a 30 nm chromatin
  fiber
• Forms only when H1 is present
• Nucleosomes are packed together to form an
  irregular 3D zig-zag structure interdigitate
  with neighbors

12
Looped Domains

• Fold 30 nm fibers into looped domains
• Loops held together by protein scaffold called
  the chromosomal scaffold
• Loops of DNA without histones and most
  non-histone proteins can be active DNA being
  transcribed
• Less tightly packed

13
Areas of Chromatin

• Tightly packed heterochromatin
• Transcriptionally inactive
• More diffusely packed euchromatin
• Actively transcribed

14
Chromosomes

• As cell prepares for cell division all
  chromatin becomes highly compacted
• Duplicated and turns into chromatids that will
  separate after mitosis

15
Mitochondria/Chloroplast DNA

• Both contain DNA, along with machinery for
  replication, transcription and translation
• No histones and circular
• Can encode some own proteins but most are from
  nuclear DNA
• Mitochondria have 37 genes and includes subunits
  of NADH dehydrogenase, cytochrome b, cytochrome c
  oxidase and ATP synthase
• Chloroplasts have larger DNA 120 genes

16
Mitochondria DNA
17
The Nucleus

• Contains cells genetic info and center for
  expression of information
• Separates from rest of cell by nuclear membrane
• Most prominent organelle

18
Structures

• Double-membrane nuclear envelope
• Separated by perinuclear space, continuous with
  ER lumen
• Inner membrane rests on nuclear lamina,
  intermembrane is continuous with ER
• Ribosomes on the surface
• Intermediate filaments anchor nucleus to plasma
  membrane or other organelles

19
Nuclear Pores

• Cylindrical channel extending thru both membranes
• Opening between cytosol and nucleoplasm (all but
  the nucleolus)
• Number of pores based on cell type and activity
• Lined with intricate protein structure nuclear
  pore complex (NPC)
• Dozens of proteins subunits in an octagon and
  protruding on both sides of membrane
• Have central core granules transporter move
  molecules thru pore
• Hub with eight spokes to hold complex in membrane
  double ring of 8 subunits
• Fibers emit from each subunit to act as trap or
  cage

20

• Must import all enzymes and proteins needed from
  cytoplasm and RNA and parts of ribosomes must
  leave the nucleus
• Pores have become specialized transporrt must
  go both ways

21
Passive Diffusion

• Pores have aqueous diffusion channels
• May have 8 separate ones per pore and one in
  transporter
• Small particles and molecules can pass thru
• Cut off of about 20,000 molecular weight
• Can move in dNTPs and NTPs in quickly
• Proteins for packaging DNA small enough to
  passively pass

22
Active Transport

• Large protein and RNA thru pores
• Proteins for replication are too large to enter
  and mRNA and protein complex (ribonucleoproteins)
  and ribosomal subunits are too large also
• Requires active transport
• Energy and specific binding proteins part of
  nuclear pore
• Cytoplasm to nucleus proteins have nuclear
  localization signal (NLS)
• Recognized by the nuclear pore
• 8-30 amino acids long, usually contains Pro, Lys
  and Arg ( charge)
• Has a maximum size that it can transport

23
Transporting

• NLS is recognized by receptor protein called
  importin moves protein to nuclear pore
• Importin-NLS transported into nucleus by
  transporter at center of NPC
• Importin associates with GTP-binding protein Ran
  and causes release of protein
• Importin-GTP-Ran is transported out of nucleus
• Importin released by GTP hydrolysis

24
Export from Nucleus

• Usually move RNA out of nucleus as a complex of
  RNA and protein
• Protein has a nuclear export signal (NES) that is
  an amino acid target
• Recognized by nuclear transport receptor proteins
  called exportins use Ran mediated GTP
  hydrolysis
• Direction is specific for type of target
• Pore can move any direction depending on whether
  importins or exportins are bound

25
Nuclear Matrix/Nuclear Lamina

• Supporting structures of nuclear membrane
• Nuclear matrix is an insoluble fibrous network
  that helps maintain the shape of the nucleus
• Organizing skeleton for chromatin fibers
• May help propel mRNA to the nuclear pore

26
Nuclear Lamina

• Thin meshwork of fibers that line the inner
  surface of the inner membrane
• Made of intermediate filaments called laminins
• Attached to proteins in the inner nuclear
  membrane
• May also be attached to chromatin fibers

27
Chromatin

• Used to be thought that chromatin was randomly
  distributed and intertwined in the nucleus
• New studies indicate that the chromatin may be in
  discreet spots called chromosome territories
  not fixed and may vary from cell to cell

28
2 Types Heterochromatin

• Constitutive highly condensed at all times in
  the cells of organism
• Simple sequence of repeated DNA
• Centromere and telomere examples
• Facultative varies with particular activities
  of the cell
• Tissue to tissue variation and cell to cell
• Specifically inactivated in a specific cell
• Low amounts in embryonic cells, more in
  differentiated cells
• May shut off blocks of genetic materia

29
Nucleolus Ribosome Formation

• 1-2 per cell but may be more
• Size based on function of cell
• High levels of protein synthesis large
• Low protein - small
• Consists of fibrils and granules
• Disappear during cell division

30
Fibrils and Granules

• Fibrils are DNA transcribed into rRNA and RNA
  part of the ribosomes
• Granules are rRNA packaged with protein into
  ribosomal subunits
• Nucleolus organizer region (NOR)
• Stretch of DNA containing multiple copies of rRNA
  genes
• Multiple NOR on multiple chromosomes
• Shortly after cell division, you see distinct
  NORs but after a short time they coalesce into 1
  NOR

31
Nucleolus as Center for Ribosomes