6. the nucleus: DNA packaging Flashcards
the human genome
- is written in 6*10^9 nucleotide bases
- contains the ~22,00 different genes
- is divided into 46 chromosomes, each an unbroken strand
- is a total of 2m long in the nucleus of 10um in diameter
Chromatin structure is only visible as ______ and ______ regions
denser, lighter
dark heterochromatin is located around the ______
nucleus periphery
active genes, in the euchromatin are located toward the _______
interior nucleus
While chromosomes haves have their own territories, there can be some overlap, so they they can share some of the ____________ machinery.
transcriptional
DNA organization level 1: histones
- DNA winds around the nucleosomes, which are composed of a group of positively charges, highly conserved proteins- histones
- DNA is held by the histones due to non-covalent bonds, especially ionic bonds between negatively charged phosphate backbone of DNA and positive charge of histone
nucleosomes
- made up of four types of histones
- two of each: H2A, H2B, H3, H4
* DNA wraps around the nucleosomes 1.8 turns or 146 nucleotide bases per nucleosome
Histone H1 (fifth type)
linker histone
- links adjacent nucleosome core particles
- a total of 168 nucleotide bases per unit
- 7.1 packing ratio, the fibre is 10nm thick
- 6-7 fold shortening
DNA organization Level 2: 30 nm fibres
spontaneous assembly of adjacent nucleosomes results in a 40:1 condensation in length, with 30 nm thick fibres
- H1 may bind adjacent nucleosomes
- N-terminal tails of H4 may extend far enough to reach other nucleosome histones
DNA organization level 3: looped domains
- the 30 nm fibres gather into supercoiled loops
- loops are tethered to protein nuclear scaffold
- cohesin- ring shaped protein may maintain the loops
- DNA strands associate with nuclear matrix
- form loops of 20,000 to 100,000 bases
- AT-rich domains on DNA form MARs (matrix associated region)
- includes alpha satellite and other non-coding DNA
MARs
DNA sequence that has affinity for nuclear matrix; note that DNA loop can be moved to facilitate transcription
Scaffold proteins includes the following:
- nuclear matrix
- topoisomerase II
- insulator protein
- nuclear lamina
nuclear matrix
- a protein fibre framework
- major organizing structure for RNA polymerase (makes messenger RNA), RNA processing, DNA replication
Topoisomerase II
-an untangling protein (for unwinding DNA)
insulator protein
keeps loops separate
nuclear lamina
lamins bind telomeres and alpha satellite DNA of the centromere
Nuclear scaffold can be observed by
digesting away all DNA and removing all histones
DNA organizing Level 4: mitotic chromosomes (a 10,000-fold decrease in length)
- also have looped domain structure
- no nucleus when chromosomes in this state
- DNA loops on Condensin protein
- more compact than interphase chromatin
- phosphorylation by MPF
- centromere is a site of extreme condensation
- contains alpha-satellite DNA
- binds to proteins, including the kinetochore
Alpha satellite DNA is comprised of _____ nucleotide repeats
171
histone code
- DNA is frequently being transcribed and duplicated
- Necessary machinery has to work around nucleosomes
- the cell makes DNA more or less accessible to other proteins by modifying histones
- mechanisms of Histone modification
- replacement with modified types of histone
- chemical modification of the histones
Modified types of histones
- H2A.X
- H2A.Z and H33.3
- CNEP-A (version of H3)
- macroH2A
H2A.X
DNA being repaired
H2A.Z
DNA transcribed by RNA polymerase II
CNEP-A
version of H3
on the centromere, kinetochore assembly
macroH2A
Barr body
keeps chromatin condensed and inactive
What does the phosphorylating?
ATM
Acetylated histones
looser DNA binding
HDAC- histone deacetylse (removes acetyl group)
HAT- histone acetyl transferase
Phosphorylated histones
can increase or decrease DNA winding on histones
methylated histones
- alter the accessibility of DNA to processing enzymes
- alter binding to organizational structures such as nuclear matrix
- often tighter DNA binding
- eg methylation of H3 leads to HP1 binding, which helps form heterochromatin
euchromatin
open, dispersed, potentially active
heterochromatin
during interphase about 10% of the chromatin remains in a compact state similar to the mitotic chromosome; this DNA is not transcribed or translated
Constitutive heterochromatin
condensed at all times
includes:
1) AT-rich satellite DNA at the centromere;
2) the telomeres (ends of the chromosomes)
facultative heterochromatin
transient condensation, contains temporarily inactive genes
In humans, gender is determined by the sex chromosomes:
females: two x chromosomes
males: one x and one y chromosome
x and y chromosomes look very different:
almost all the gens on the x have no counterpart on the y
To ensure that males and females have equal levels of gene expression derived from the x chromosome what happens?
one x in every female cell is inactivated
which x is apparently random
inactivation of an x chromosome requires a gene on that chromosomes called ______
XIST
XIST
encodes a large molecule of RNA
XIST RNA
- accumulates along the x chromosome containing the active XIST gene and proceeds to inactivate almost all of the other genes on that chromosome
- does not travel over to the other x chromosome in the nucleus
Barr bodies
are inactive x chromosomes painted with XIST RNA
inactive x chromosome is
neatly straight and organized
How is the Barr body condensed?
- XIST RNA induces several later events (by recruiting various enzymes):
- deacetylation of H3
- methylation of H3
- Ubiquitination of H2A
- H2A is replaced with macroH2A
- all these changes are undone in egg cells
what does random inactivation of x result in?
mosaic patterns of gene expression
ex. calico cats: always female ( except in rare XXY males )
*x chromosome has gene for coat colour
*the gene can encode either a black pigment of orange
*since males have only one x, they can be either black or orange, not both
-the X chromosome inactivation in females is random, so different patches of epidermal cells can have different colour genes
(the gene for white coat is on another chromosome)
interphase chromatin
- storing DNA is an exercise in organization
- although not easily seen in interphase, each unwound chromosome has a defined territory within the nucleus
* held in place on the nuclear matrix and nuclear laimina, ie. protein frameworks
* where each chromosome is positioned can affect the expression of its genes
