1
Chromatin Structure
Chromatin
First
DNA
g g
“packaging”
a basic discussion of protein-nucleic acid binding
Questions to answer:
How do proteins bind DNA / RNA ?
How do proteins recognize a specific nucleic acid sequence?
forces / interactions involved in protein-nucleic acid binding
force / interaction
parts of protein/nucleic acid
involved in binding
What forces are most important for sequence-specific interaction?
2
(helps stabilize / strengthen)
(some ring stacking with specific
bases / specific A.A. side chains
precisely positioned)
increasing
importance
for sequencespecific
p
binding
g
(the defined length and linearity
of the hydrogen-bond requires
very specific positioning of
defined nucleotide and amino
acid sequences)
Note: all three forces are likely to be utilized in sequencespecific interaction but their importance varies
Packaging DNA in Chromatin Structure
nucleic acid binding proteins
eukaryotic chromatin
why package DNA?
eukaryotic cell
(requires compaction
of about 10,000 fold)
two types of chromatin proteins
1. basic proteins (high pI)
(simple composition)
2. acidic proteins (low pI)
(complex composition)
chromatin composition (mass ratio)
DNA
3
: histones : NHCP
packaging
proteins
replication/transcription
splicing/regulatory proteins
Hi t
Histones
histone
mol wt (kD) amino acids
A.A. comp.
Lys/Arg
23
213
Lys rich (29%)
21
13.9
129
Lys rich (11%)
1.2
13.7
125
Lys rich (16%)
2.5
15.3
135
Arg rich (14%)
0.7
11 2
11.2
102
A rich
Arg
i h (14%)
08
0.8
(Note: H2A-H2B, H3-H4 pairs and unique H1 have structural significance.)
What can you say about the histones as a class of proteins??
1. Proteins are defined by chromatin extraction procedure.
extracted/soluble
t t d/ l bl in
i 0.1
0 1 M HCl (high
(hi h pI)
I)
proteins remaining/insoluble in 0.1 M HCl
4
2. Histones as a group:
NH2
1/3
2/3
COOH
3. Specific amino acids are modified
Lys
Lys / Arg / His
Ser
affects
DNA
binding
4. Evolutionarily highly conserved primary sequence
H3-H4 most conserved
H4: cow vs pea
2 A.A. differences
H2A-H2B highly conserved
H1 least conserved
H3: cow vs pea
4 A.A. differences
experimental approach
examine chromatin structure
(original approach to examine
chromatin structure)
(non-specific
S.S. and D.S.
nuclease)
determines sites of
nuclease accessability
on folded chromatin
analytical techniques
size + amount of
DNA-protein complexes
size and composition
of digested DNA
protein composition
sucrose gradient analysis of digested chromatin
What does this digestion
pattern indicate??
A260
S
5
What does this digestion
pattern indicate??
6
A260
S
agarose gel electrophoresis of DNA from digested chromatin
0
1
2
3
Whatt does
Wh
d
this
thi digestion
di
ti
pattern indicate??
4
5
6
7
electron microscopic analysis of chromatin
7
undigested chromatin
(spread on a microscope slide)
analysis of sucrose gradient peaks
DNA length
EM structure
gradient peak
11S
15S
18 S
monomer bead (11S and 200 bp DNA)
SDS polyacrylamide gel analysis of 11S protein composition
histone protein ratio
H1 : H3 : H2A : H2B : H4
What is the number of the
different histone molecules
in a single nucleosome??
+
further digestion of chromatin
3 stages of digestion
8
H1
(200 bp DNA + 5 histones)
(160 bp DNA + 5 histones)
H3
H2A
H2B
H4
extensive
digestion
140 bp DNA +
4 histones (H2A, H2B, H3,H4)
minus H1
core
11S and
“pause” complex
electrophoretic analysis of DNA
11 S
p
a
u
s
e
c
o
r
e
extensive
digestion
What does this digestion
pattern tell you about
nucleosome structure??
1.
2.
3.
4.
Basic structural organization of a nucleosome
nucleosome
core
9
nucleosome
core
(H2A-H2B) x 2
(H3-H4)
(H3
H4) x 2
(H2A-H2B) x 2
(H3-H4) x 2
What is the order of
digestion accessibility
f sites
for
it A,
A B,
B C,
C & D?
DNA path
A
B
A
C
A
D
Structure of the histone nucleosome core
(H3 H4 di
(H3-H4
dimer)) x 2
(H2A-H2B dimer) x 2
H2A
H2B H3-H4
H2A
x 2 H2B
2 L.H. DNA turns
around this core
10
PNAS (1993) 90:10490
H3 = green
H4 = white
H2A = light blue
H2B = d
dark
k bl
blue
histone octamer forms
the nucleosome core
H2A
H2B H3-H4
H2A
x 2 H2B
Two full turns of the
DNA is wrapped around
the histone octamer
core
Histone H1 is not present
in the nucleosome core
11
Nature ((1997)) 389:233
This is only a half
nucleosome core
possessing only one
H2A-H2B dimer and
one H3-H4 dimer with
one full turn of the DNA
What is the
significance of
the N-terminal
histone tails??
12
Higher order chromatin folding
additional nucleosome folding
(5-7 fold compaction)
helix of nucleosomes
=
H1 involvement
path of DNA
(2 L.H. turns / nucleosome)
important for nucleosome spacing
and solenoid formation
higher order folding
(large loops of chromatin)
still poorly understood
(10,000 - fold compaction needed)