Transcript Enzyme Mechanisms - Illinois Institute of Technology

Nucleic Acid
Structure II
Andy Howard
Introductory Biochemistry
9 October 2008
Biochemistry: Nucleic Acid Struct II
10/09/08
What we’ll discuss
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Folding kinetics
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Supercoils
Nucleosomes
Chromatin and chromosomes
Lab synthesis of genes
tRNA & rRNA structure
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Getting from B to Z
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Can be accomplished without breaking
bonds
… even though purines have their
glycosidic bonds flipped (anti -> syn) and
the pyrimidines are flipped altogether!
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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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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 by
Moudrianakis, and 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 the 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
Histone
#lys, #arg
Mr, kDa
Copies per
Nucleosome
H1
59, 3
21.2
H2A
13, 13
14.1
1 (not in
bead)
2 (in bead)
H2B
20, 8
13.9
2 (in bead)
H3
13, 17
15.1
2 (in bead)
H4
11, 14
11.4
2 (in bead)
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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
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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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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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Synthesizing nucleic acids
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Laboratory synthesis of nucleic acids
requires complex strategies
Functional groups on the monomeric
units are reactive and must be blocked
Correct phosphodiester linkages must
be made
Recovery at each step must high!
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Solid Phase
Oligonucleotide
Synthesis
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Dimethoxytrityl group blocks the 5'-OH of the
first nucleoside while it is linked to a solid
support by the 3'-OH
Step 1: Detritylation by trichloroacetic acid
exposes the 5'-OH
Step 2: In coupling reaction, second base is
added as a nucleoside phosphoramidate
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Figure 11.29
Solid phase oligonucleotide
synthesis. The four-step cycle
starts with the first base in
nucleoside form (N-1) attached by
its 3'-OH group to an insoluble,
inert resin or matrix, typically either
controlled pore glass (CPG) or
silica beads. Its 5'-OH is blocked
with a dimethoxytrityl (DMTr) group
(a). If the base has reactive -NH2
functions, as in A, G, or C, then Nbenzoyl or N-isobutyryl derivatives
are used to prevent their reaction
(b). In step 1, the DMTr protecting
group is removed by trichloroacetic
acid treatment. Step 2 is the
coupling step: the second base (N2) is added in the form of a
nucleoside phosphoramidite
derivative whose 5'-OH bears a
DMTr blocking group so it cannot
polymerize with itself (c).
Solid Phase Synthesis
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Step 3: capping with acetic anhydride
blocks unreacted 5’-OHs of N-1 from further
reaction
Step 4: Phosphite linkage between N-1 and
N-2 is reactive and is oxidized by aqueous
iodine to form the desired, and more stable,
phosphate group
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Activation of the
phosphoramidate
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Secondary and Tertiary
Structure of RNA
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Transfer RNA
Extensive H-bonding creates four double
helical domains, three capped by loops, one
by a stem
Only one tRNA structure (alone) is known
Phenylalanine tRNA is "L-shaped"
Many non-canonical base pairs found in tRNA
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tRNA
structure:
overview
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Amino acid
linkage to
acceptor stem
Amino acids are linked to the 3'OH end of tRNA molecules by
an ester bond formed between
the carboxyl group of the
amino acid and the 3'-OH of
the terminal ribose of the
tRNA.
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Yeast phetRNA
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Note
nonstandard
bases and
cloverleaf
structure
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