Download Organization and Synthesis of DNA

Organization of DNA
Andy Howard
Introductory Biochemistry
12 October 2010
Biochem: Nucleic Acid Structure II
10/12/2010
What we’ll discuss
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Restriction Enzymes
Review of A,B,Z DNA
Intercalation
Denaturation and
renaturation of DNA
DNA density
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DNA tertiary structure
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Review of supercoiling
Gyrases
Nucleosomes
Higher levels
Bacterial organization
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Restriction Endonucleases
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Evolve in bacteria as antiviral tools
“Restriction” because they restrict the
incorporation of foreign DNA into the bacterial
chromosome
Recognize and bind to specific palindromic DNA
sequences and cleave them
Self-cleavage avoided by methylation
Types I, II, III: II is most important
I and III have inherent methylase activity; II has
methylase activity in an attendant enzyme
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What do we mean
by palindromic?
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In ordinary language, it means a phrase that reads
the same forward and back:
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Madam, I’m Adam. (Genesis 3:20)
Eve, man, am Eve.
Sex at noon taxes.
Able was I ere I saw Elba. (Napoleon)
A man, a plan, a canal: Panama!
(T. Roosevelt)
With DNA it means the double-stranded sequence
is identical on both strands
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Quirky math problem
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Numbers can be palindromic:
484, 1331, 727, 595…
Some numbers that are palindromic have
squares that are palindromic…
222 = 484, 1212 = 14641, . . .
Question: if a number is perfect square and
a palindrome, is its square root a
palindrome? (answer will be given orally)
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Palindromic DNA
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Example: G-A-A-T-T-C
Single strand isn’t symmetric: but the
combination with the complementary strand
is:
G-A-A-T-T-C
C-T-T-A-A-G
These kinds of sequences are the
recognition sites for restriction
endonucleases. This particular
hexanucleotide is the recognition sequence
for EcoRI.
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Cleavages by restriction
endonucleases
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Breaks can be
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cohesive (if off-center within the sequence) or
non-cohesive (blunt) (if they’re at the center)
EcoRI leaves staggered 5’-termini: cleaves
between initial G and A
PstI cleaves CTGCAG between A and G, so it
leaves staggered 3’-termini
BalI cleaves TGGCCA in the middle: blunt!
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iClicker question
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1. Which of the following is a potential
restriction site?
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(a) ACTTCA
(b) AGCGCT
(c) TGGCCT
(d) AACCGG
(e) none of the above.
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Example for E.coli
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5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’
Cleaves G-A on top, A-G on bottom:
5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’
Protruding 5’ ends:
5’-N-N-N-N-G
A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A
G-N-N-N-N-5’
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How often?
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4 types of bases
So a recognition site that is 4 bases long
will occur once every 44 = 256 bases on
either strand, on average
6-base site: every 46= 4096 bases, which is
roughly one gene’s worth
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EcoRI structure
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Dimeric structure
enables recognition of
palindromic sequence
 sandwich in each
monomer
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
EcoRI pre-recognition
complex
PDB 1CL8
57 kDa dimer + DNA
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The biology problem
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How does the bacterium mark its own DNA
so that it does replicate its own DNA but not
the foreign DNA?
Answer: by methylating specific bases in its
DNA prior to replication
Unmethylated DNA from foreign source
gets cleaved by restriction endonuclease
Only the methylated DNA survives to be
replicated
Most methylations are of A & G,
but sometimes C gets it too
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How this works
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When an unmethylated specific
sequence appears in the DNA, the
enzyme cleaves it
When the corresponding methylated
sequence appears, it doesn’t get cleaved
and remains available for replication
The restriction endonucleases only bind
to palindromic sequences
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Methylases
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A typical bacterium
protects its own DNA
against cleavage by
its restriction
endonucleases by
methylating a base in
the restriction site
Methylating agent is
generally Sadenosylmethionine
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
HhaI methyltransferase
PDB 1SVU
2.66Å; 72 kDa dimer
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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Use of restriction enzymes
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Nature made these to protect bacteria; we
use them to cleave DNA in analyzable ways
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Similar to proteolytic digestion of proteins
Having a variety of nucleases means we can get
fragments in multiple ways
We can amplify our DNA first
Can also be used in synthesis of inserts that
we can incorporate into plasmids that enable
us to make appropriate DNA molecules in
bacteria
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Summaries of A, B, Z DNA
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DNA is dynamic
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Don’t think of these diagrams as static
The H-bonds stretch and the torsions
allow some rotations, so the ropes can
form roughly spherical shapes when not
constrained by histones
Shape is sequence-dependent, which
influences protein-DNA interactions
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Intercalating agents
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Generally: aromatic compounds that can
form -stack interactions with bases
Bases must be forced apart to fit them in
Results in an almost ladderlike structure
for the sugar-phosphate backbone locally
Conclusion: it must be easy to do local
unwinding to get those in!
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Instances
of intercalators
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Denaturing and Renaturing DNA
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See Figure 11.17
When DNA is heated to 80+ degrees
Celsius, its UV absorbance increases by
30-40%
This hyperchromic shift reflects the
unwinding of the DNA double helix
Stacked base pairs in native DNA absorb
less light
When T is lowered, the absorbance drops,
reflecting the re-establishment of stacking
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Heat denaturation
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Figure 11.14
Heat denaturation of DNA from various sources, so-called
melting curves. The midpoint of the melting curve is
defined as the melting temperature, Tm.
(From Marmur, J., 1959. Nature 183:1427–1429.)
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GC content
vs. melting
temp
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High salt and
no chelators
raises the
melting
temperature
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How else can we melt DNA?
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High pH deprotonates the bases so the Hbonds disappear
Low pH hyper-protonates the bases so the
H-bonds disappear
Alkalai is better: it doesn’t break the
glycosidic linkages
Urea, formamide make better H-bonds
than the DNA itself so they denature DNA
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What happens if we
separate the strands?
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We can renature the DNA into a double
helix
Requires re-association of 2 strands:
reannealing
The realignment can go wrong
Association is 2nd-order, zippering is first
order and therefore faster
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Steps in denaturation
and renaturation
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Rate depends on complexity
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The more complex DNA is, the longer it
takes for nucleation of renaturation to
occur
“Complex” can mean “large”, but
complexity is influenced by sequence
randomness: poly(AT) is faster than a
random sequence
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Second-order kinetics
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Rate of association: -dc/dt = k2c2
Boundary condition is fully denatured
concentration c0 at time t=0:
c / c0 = (1+k2c0t)-1
Half time is t1/2 = (k2c0)-1
Routine depiction: plot c0t vs. fraction
reassociated (c /c0) and find the halfway
point.
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Typical c0t curves
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Hybrid
duplexes
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We can associate DNA
from 2 species
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Closer relatives hybridize
better
Can be probed one gene
at a time
DNA-RNA hybrids can
be used to fish out
appropriate RNA
molecules
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GC-rich DNA is denser
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DNA is denser than RNA or protein,
period, because it can coil up so
compactly
Therefore density-gradient centrifugation
separates DNA from other cellular
macromolecules
GC-rich DNA is 3% denser than AT-rich
Can be used as a quick measure of GC
content
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Density
as
function
of GC
content
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Tertiary Structure of DNA
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In duplex DNA, ten bp per turn of helix
Circular DNA sometimes has more or less
than 10 bp per turn - a supercoiled state
Enzymes called topoisomerases or gyrases
can introduce or remove supercoils
Cruciforms occur in palindromic regions of
DNA
Negative supercoiling may promote
cruciforms
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DNA is wound
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Standard is one winding per helical
turn, i.e. 1 winding per 10 bp
Fewer coils or more coils can happen:
This introduces stresses that favors
unwinding
Both underwound and overwound
DNA compact the DNA so it sediments
faster than relaxed DNA
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Linking, twists, and writhe
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T=Twist=number of helical turns
W=Writhe=number of supercoils
L=T+W = Linking number is constant
unless you break covalent bonds
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Examples
with a tube
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How this works with real DNA
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How gyrases
work
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Enzyme cuts the
DNA and lets the
DNA pass through
itself
Then the enzyme
religates the DNA
Can introduce new
supercoils or take
away old ones
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Typical gyrase
action
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Takes W=0
circular DNA and
supercoils it to
W=-4
This then relaxes
a little by
disrupting some
base-pairs to
make ssDNA
bubbles
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Superhelix density
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Compare L for real DNA to what it would
be if it were relaxed (W=0):
That’s L = L - L0
Sometimes we want
 = superhelix density
= specific linking difference = L / L0
Natural circular DNA always has  < 0
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 < 0 and spools
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The strain in  < 0 DNA can be alleviated
by wrapping the DNA around protein spool
That’s part of what stabilizes nucleosomes
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Cruciform DNA
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Cross-shaped structures arise from
palindromic structures, including
interrupted palindromes like this
example
These are less stable than regular
duplexes but they are common,
and they do create recognition sites
for DNA-binding proteins, including
restriction enzymes
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Cruciform DNA example
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Eukaryotic chromosome structure
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Human DNA’s total length is ~2 meters!
This must be packaged into a nucleus that
is about 5 micrometers in diameter
This represents a compression of more
than 100,000!
It is made possible by wrapping the DNA
around protein spools called nucleosomes
and then packing these in helical filaments
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Chromatin
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Discovered long before we
understood molecular
biology
Seen to be banded objects
in nuclei of stained
eukaryotic cells
In resting cell it exists as
long slender threads, 30
nm diameter
From answers.com
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Squishing the DNA
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If the double helix were fully extended,
the largest human chromosome
(2.4*108bp) would be 2.4*108 *0.33nm ~
0.8*108nm=80 mm;
much bigger than the cell!
So we have to coil it up a lot to make it fit.
Longest chromosome is 10µm long
So the packing ratio is 80mm/10µm =
8000
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Chromosome
structure:
levels
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Each of the
first 4 levels
compacts DNA
by a factor of
6-20; those
multiply up to
> 104
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Nucleosome Structure
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Chromatin, the nucleoprotein
complex, consists of histones and
nonhistone chromosomal proteins
Histone octamer structure has
been solved
without DNA: Moudrianakis, 1991
with DNA by Richmond
Nonhistone proteins are regulators
of gene expression
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Histone types
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H2a, H2b, H3, H4 make up core particle:
two copies of each, so: octamer
All histones are KR-rich, small proteins
H1 associates with the regions between
the nucleosomes
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Histones: table 11.2, plus…
Histone
#lys,
#arg
# Mr,
acidic kDa
H1
59, 3
10 21.2
H2A
13, 13
9 14.1
2 (in bead)
H2B
20, 8
10 13.9
2 (in bead)
H3
13, 17
11 15.1
2 (in bead)
H4
11, 14
7 11.4
2 (in bead)
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Copies per
Nucleosome
1 (not in bead)
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Unfolded chromatin
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Treat chromatin with low ionic strength;
that disrupts higher level interactions so
the individual nucleosomes are strung
out relative to one another like beads on
a string
Image
courtesy
U. Maine
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Nucleosome core particle
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Half the core
particle
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Note that DNA
isn’t really
circular: it’s a
series of straight
sections followed
by bends (like the
Advanced Photon
Source ring!)
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Histones, continued
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Individual nucleosomes
attach via histone H1 to
seal the ends of the turns
on the core and organize
40-60bp of DNA linking
consecutive nucleosomes
N-terminal tails of H3 &
H4 are accessible
K, S get post-translational
modifications, particularly
K-acetylation
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O
HN
Histone
deactivation
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ONH3+
O
acylated lysine
Histones interact with DNA via
+charges on lys and arg residues.
If we neutralize those charges by
acetylation, the histones don’t bind as
tightly to the DNA
Carefully-timed enzymatic control of
histone acetylation is a crucial element
in DNA organization
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CoASH
Histone
acetylation
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Active histone + Acetyl CoA
 inactive (acetylated)
histone + CoASH
Without the positive
charges, the affinity for DNA
goes down
Histone H1
PDB 1GHC
8.3 kDa monomer
Chicken
Histone
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acetyltransferase
PDB 1QSO
66 kDa
tetramer
yeast
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Histone
deacetylation
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Type III deacetylases use
a non-trivial reaction:
Prot-lys-NAc + NAD+ 
Prot-lys-NH3+ + nicotinamide +
2’-O-acetyl-ADP-ribose
Part of the NAD salvage
pathway
Histone/protein deacetylase +
histone H4 active peptide
PDB 1SZD; 34 kDa “heterodimer”
yeast
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Other histone PTM
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Histones can be post-translationally
modified in other ways as well
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Methylation: e.g. lysines 4,27 of H3
Phosphorylation: H2A phosphorylated at
several sites near “hinge”
These are correlated with acetylation and
play a role in folding and function
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Nucleosome
structure
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Core octamer is two molecules
each of H2A, H2B, H3, H4
Typically wraps around
~200bp of DNA
DNA between
nucleosomes is ~54 bp long
H1 binds to linker and to core
particle; but in beads-on-a-string
structure, it’s often absent
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How much does this coil up?
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200 bp extended would be about 50nm
The width of the core-particle disk is 5nm
So this is a tenfold reduction
Nucleosomal organization corresponds to
negative supercoiling
… so DNA ends up supercoiled when we
take away the histones
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Courtesy answers.com
Next level of
organization
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H1 interacts with
DNA along linker
region
Individual histones
spiral along to form
30 nm fiber
See fig.19.25
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Courtesy
Johns
Hopkins
Univ
p. 60 of 63
Even higher…
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The 30nm fibers are attached to
an RNA-protein scaffold that
holds the 30nm fibers in large
loops
Typical chromosome has ~200
loops
Loops are attached to scaffold at
their base
Ends can rotate so it can be
supercoiled
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What about prokaryotes?
No actual histones
 Histone-like proteins (HLPs)
involved
 Bacterial DNA attached to scaffold
in large loops (~100kb)
 This makes a nucleoid
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How many loops in bacteria?
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Typical bacterial genome (E.coli) has
3000 open reading frames ~ 3000
genes.
Assume 500 amino acids per protein =
1500 bases per gene (ignores
transcriptional elements)
Then genome is 1500 bp/gene * 3000
genes = 4.5*106 base-pairs
That’s (4.5*106 bp)/(1*105 bp/loop) = 45
loops
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