Transcript lecture6

Homework (Wednesday, 9/27/06, Due on Wednesday, 10/4/06)
1. Show the molecular orbitals of P15.
2. Calculate the exact molecular weights of each of the four
standard nucleotides (A, T, G, and C) in DNA molecules?
3. pBR322 is a circular double-stranded plasmid of 4363 nucleotides.
Figure out 1) how many picomoles are 1 microgram of the plasmid?
2) How many micrograms are 1 pmol of the plasmid? 3) How much
are the weights of two single-stranded circular pBBR322, respectively?
4. Draw diagrams to show all possible base-pairings of adenine
and thymine.
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DNA structure:
Secondary Structure: alternative conformations
Minor groove
The minor groove is generated by
the smaller angular distance between sugars.
Major groove
major groove:
The larger of the two grooves that spiral
around the surface of the B-form of DNA.
DNA structure:
Secondary Structure: alternative conformations
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DNA structure:
Secondary Structure: alternative conformations
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Minor groove
Minor groove
B-form
* Most common DNA conformation in vivo
Major groove
* Narrower, more elongated helix than A.
* Wide major groove easily accessible to proteins
* Narrow minor groove
* Favored conformation at high water concentrations
(hydration of minor groove seems to favor B-form)
* Base pairs nearly perpendicular to helix axis
* Sugar pucker C2'-endo
A-form
* Most RNA and RNA-DNA duplex in this form
* shorter, wider helix than B.
* deep, narrow major groove not easily accessible to proteins
* wide, shallow minor groove accessible to proteins,
but lower information content than major groove.
* favored conformation at low water concentrations
* base pairs tilted to helix axis
* Sugar pucker C3'-endo (in RNA 2'-OH inhibits
C2'-endo conformation)
Minor groove
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DNA structure:
Secondary Structure: alternative conformations
Minor groove
Z-form
* Helix has left-handed sense
* Can be formed in vivo, given proper sequence
and superhelical tension,
but function remains obscure.
* Narrower, more elongated helix than A or B.
* Major "groove" not really groove
* Narrow minor groove
* Conformation favored by high salt concentrations,
some base substitutions, but requires
alternating purine-pyrimidine sequence.
* N2-amino of G H-bonds to 5' PO: explains
slow exchange of proton, need for G purine.
* Base pairs nearly perpendicular to helix axis
* GpC repeat, not single base-pair
H
o P-P distances: vary for GpC and CpG
o GpC stack: good base overlap
o CpG: less overlap.
* Zigzag backbone due to C sugar conformation
compensating for G glycosidic bond conformation
* Conformations:
o G; syn, C2'-endo
o C; anti, C3'-endo
Major groove
H
H
Syn-deoxyadenosine
H
H
Anti-deoxyadenosine
DNA structure:
Secondary Structure: alternative conformations
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In Z-DNA and Z-RNA the (desoxy)ribo-phophate backbone forms
a left-handed, zigzag helix where the glycosidic bonds between
base and sugar alternate between the syn- and anti-conformation
and the purines are tilted to the outside.
In contrast, in B-DNA and A-RNA the backbone forms a smooth
right-handed helix and all nucleotides are in the anti-conformation
facing towards the inside of the helix. At the boundary between
B-DNA and Z-DNA, a B-Z junction is formed in which a base pair
is broken and the bases are extruded on each side.
Cellular functions of Z-DNA:
1. Z-DNA can either enhance or repress promoter activity.
2. Z-DNA and Z-RNA can be bound by Z-DNA binding proteins (ZBPs),
modulating immune functions.
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DNA structure:
Secondary Structure: visualizing DNA by X-Ray crystallography
An average chemical bond has an average bond of 1.5 angstroms.
X-ray is photons with a wavelength ranging from 0.05 to 100 angstroms.
Filaments
Detector
Electrons
A crystal
Diffracted X-Ray
Metal target
(Cu)
Primary X-ray
(CuKα)
or from synchrotrons
Grow crystals by hanging drop setup
DNA Secondary structure:
Secondary Structure: visualizing DNA by X-Ray crystallography
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A synchrotron is a particular type of cyclic particle accelerator in which
the magnetic field (to turn the particles so they circulate) and the electric
field (to accelerate the particles) are carefully synchronized with the
traveling particle beam.
Currently, the highest energy synchrotron in the world is the Tevatron, at the Fermi National
Accelerator Laboratory, in the United States. It accelerates protons and antiprotons to slightly
less than 1 TeV of kinetic energy and collides them together. The Large Hadron Collider (LHC),
which is being built at the European Laboratory for High Energy Physics (CERN), will have
roughly seven times this energy, and is scheduled to turn on in 2007. It is being built in the
27 km tunnel which formerly housed the Large Electron Positron (LEP) collider, so it will
maintain the claim as the largest scientific device ever built. The LHC will also accelerate
heavy ions (such as Lead) up to an energy of 1150 TeV.
The largest device of this type seriously proposed was the Superconducting Super Collider (SSC),
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Structure biologists use synchrotron radiation (synchrotron X-rays)
to illuminate the crystals of biological samples.
Advantages: 1) Small crystals; 2) Short exposures
DNA Secondary structure:
Secondary Structure: visualizing DNA by X-Ray crystallography
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In 1979, Rich and co-workers at MIT accidentally grew a crystal of ZDNA[1]. This was the first crystal structure of any form of DNA. After
26 years of attempts, Rich et al. finally crystallized the junction box of
B- and Z-DNA. Their results were published in an October 2005 Nature
journal[2]. Whenever Z-DNA forms, there must be two junction boxes
that allow the flip back to the canonical B-form of DNA.
[1]. Wang AHJ, Quigley GJ, Kolpak FJ, Crawford JL, van Boom JH, Van der Marel G,
and Rich A (1979). Molecular structure of a left-handed double helical DNA fragment
at atomic resolution. Nature (London) 282:680-686.
[2]. Ha SC, Lowenhaupt K, Rich A, Kim YG, and Kim KK (2005). Crystal structure of
a junction between B-DNA and Z-DNA reveals two extruded bases. Nature 437:1183-1186.
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DNA structure:
Secondary Structure: comparisons of alternative forms
Properties of different helical forms
Geometry attribute
Helix sense
A-form
B-form
Z-form
right-handed
right-handed
left-handed
Repeating unit
1 bp
1 bp
2 bp
33.6°
35.9° (±4.2°)
60°/2
Mean bp/turn
10.7
10.0 (±1.2)
12
Inclination of bp to axis
+19°
-1.2° (±4.1°)
-9°
Rise/bp along axis
0.23 nm
0.332 nm (±0.019nm)
0.38 nm
Pitch/turn of helix
2.46 nm
3.32 nm (±0.19nm)
4.56 nm
+18°
+16° ( ±7°)
0°
Rotation/bp
Mean propeller twist
Glycosyl angle
anti
anti
C: anti,
G: syn
Sugar pucker
C3'-endo
C2'-endo
C: C2'-endo,
G: C2'-exo
Diameter
2.55 nm
2.37 nm
1.84 nm
DNA structure:
Secondary Structure: comparisons of alternative forms
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Crystallization of the luciferase D3
DNA structure:
Secondary Structure: X-ray crystallography helps resolve protein structure
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(μg)
0.5 mm
Crystals of the D3 luciferase
Tag-free D3 luciferase
Liu et al., Acta Crystallogr. D 59: 761-764, 2003.
DNA structure:
Secondary Structure: NMR helps resolve DNA structures
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Spectroscopy is the study of the interaction of electromagnetic radiation with matter.
Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study
physical, chemical, and biological properties of matter.
Spin
What is spin? Spin is a fundamental property of nature like electrical charge or mass. Spin
comes in multiples of 1/2 and can be + or -. Protons, electrons, and neutrons possess spin.
Individual unpaired electrons, protons, and neutrons each possesses a spin of 1/2.
In the deuterium atom ( 2H ), with one unpaired electron, one unpaired proton, and one
unpaired neutron, the total electronic spin = 1/2 and the total nuclear spin = 1.
When placed in a magnetic field of strength B, a particle with a net spin can absorb a
photon, of frequency . The frequency ν depends on the gyromagnetic ratio, γ of the
particle. ν = , γ B
For hydrogen, = 42.58 MHz / T.
Nuclei with Spin
The shell model for the nucleus tells us that nucleons, just like electrons, fill orbitals. When
the number of protons or neutrons equals 2, 8, 20, 28, 50, 82, and 126, orbitals are filled.
Because nucleons have spin, just like electrons do, their spin can pair up when the orbitals
are being filled and cancel out.
Energy Levels
To understand how particles with spin behave in a magnetic field, consider a proton. This
proton has the property called spin. Think of the spin of this proton as a magnetic moment
vector, causing the proton to behave like a tiny magnet with a north and south pole
DNA structure:
Secondary Structure: NMR helps resolve DNA structures
1. A spinning charge generates a
magnetic field, as shown by the
animation on the right.
The resulting spin-magnet has a
magnetic moment (μ) proportional to
the spin.
2. In the presence of an external magnetic
field (B0), two spin states exist, +1/2 and 1/2. The magnetic moment of the lower
energy +1/2 state is aligned with the
external field, but that of the higher energy
-1/2 spin state is opposed to the external
field. Note that the arrow representing the
external field points North
3. The difference in energy between the two spin states is
dependent on the external magnetic field strength, and is always
very small. The following diagram illustrates that the two spin states
have the same energy when the external field is zero, but diverge
as the field increases. At a field equal to Bx a formula for the energy
difference is given (remember I = 1/2 and μ is the magnetic moment
of the nucleus in the field).
4. For spin 1/2 nuclei the energy difference between the two spin states at a
given magnetic field strength will be proportional to their magnetic moments.
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DNA structure:
Secondary Structure: NMR helps resolve DNA structures
If the magnetic field is
smoothly increased to
2.3488 T, the hydrogen
nuclei of the water
molecules will at some
point absorb rf energy
and a resonance signal
will appear.
TROSY (transverse relaxation-optimized
spectroscopy) makes use of the fact that
cancellation of transverse relaxation
effects can be achieved for one of the four
multiplet components observed for 15N1H moieties in 15N-labeled proteins, and
TROSY exclusively observes this narrow
component [3]. Theory predicts that the
extent of this cancellation effect is
dependent on the polarizing magnetic field
B.: at 1H NMR frequencies in the range
900-1000 MHz it may be nearly
complete[3, 4], but TROSY yields
significantly narrower spectral linewidths
and improved sensitivity for observation of
15 N-1H groups already at 750 MHz, as
was recently verified using experiments
with an oligomeric protein of molecular
weight 110 000.
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DNA structure:
Secondary Structure: Base Stacking
The electrons are actually shared in such a pattern, so that
the density between all the atoms is close to 1.5 bonds, and
the p orbitals overlap to form a system called a pi orbital.
The simple benzene molecule, where all six atoms are equivalent,
is pictured at the right. The electron density is uniformly distributed
around the ring, and one component of the pi-electron system is
the sum of the electron lobes seen here. The sum of all pi-orbitals
is symmetric above and below the ring. Rings that stack share
pi-electrons, resulting in a favorable configuration.
Stacks of planer systems with multiple conjugated rings are favored even more,
thus the purines, particularly guanine, have high stacking energies.
Base Stacking:
1. When the bases are stacked,
the charges contribute to the stacking energy.
2. the electrostatic field of one base induce
attractive, asymmetrical electronic distributions
in the electron distribution of the other base.
3. Spontaneous fluctuations in charge occur
in each base and induce dipoles in the other.
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DNA structure:
Secondary Structure: Triple helical DNA
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Coordination of transition metal cations to purine nucleotides in the third strand may cause polarization of the
base charges, eventually leading to stronger hydrogen bonds with the complementary purine nucleotides in one
strand of the target DNA duplex, thereby strengthening the triplex structure.
Another source of triplex stability is related to stretches of cationic amino acid residues in proteins.
DNA structure:
Secondary Structure: Four-stranded DNA
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DNA structure:
Non-helical secondary structures: non-helical , palindrome, hairpin, and cruciform
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stabilized by negative DNA
supercoiling is a cruciform in
which an inverted repeat
nucleotide sequence rearranges
from a fully double-stranded
structure into two base-paired
hairpins.
Images of 106 bp inverted repeat
generated by atomic force microscope.
(i) low salt and low superhelicity;
(ii) (ii) high Salt and high superhelicity.