DNA&RNA.ppt

13. Introduction to DNA and RNA structure
View the “The DNA Story” movie at one of these times:
Thursday, Feb. 8 at 8:30 a.m. in Kane 110
Friday, Feb. 9 at 10:30 a.m. in Kane 210
Friday, Feb. 9 at 2:30 p.m. in Kane 110

 2’-deoxyribose sugars
 Phosphodiester linkages
 Directional chain (5’ to 3’)
 4 Bases
purines: adenine & guanine
pyrimidines: cytosine & thymine
DNA is a polymer of
2’-deoxyribonucleotides
GCTAp
5’ end
3’ end
C
G
T
A
HO-CH2
O
H2N-C
C
C
HN
N
N
CH
C
O
N
O
O
O P O CH2
O
O
C
N
N
CH
C
CH
NH2
NH2
C
C
N
N
N
CH
C
N
HC
O
O
O P O CH2
O
O-PO3
2
O
O
O P O CH2
O
N
C
C
O
HN
CH
C
O
CH3
1’
2’
3’
4’
5’
3’

RNA is a polymer of
ribonucleotides
 ribose sugars
 Phosphodiester linkages
 Directional chain (5’ to 3’)
 4 Bases
purines: adenine & guanine
pyrimidines: cytosine & uracil
GCUAp
C
G
U
A
5’ end
3’ end
1’
2’
3’
4’
5’
3’
OH
HO-CH2
O
H2N-C
C
C
HN
N
N
CH
C
O
N
O
O
O P O CH2
O
O
C
N
N
CH
C
CH
NH2
OH
O
O
O P O CH2
O
N
CH
C
O
HN
CH
C
O
OH
NH2
C
C
N
N
N
CH
C
N
HC
O
O
O P O CH2
O
O-PO3
2
OH

RNA is easily hydrolyzed under alkaline conditions
The reaction proceeds through a 2’,3’-cyclic monophosphate intermediate.
Enzymatic hydrolysis of RNA by RNase proceeds through a similar intermediate.
Because DNA lacks the 2’-OH group, it is stable under alkaline conditions.
.
.
.
.
O P O-CH2
O
O
O
N
OH
O
O P O CH2
O
O
N
OH
O
O P O
O
...
OH
O
N
OH
O
O P O
O
...
HOCH2
O
.
.
.
.
O P O-CH2
O
O
N
O
O
P
O
O
H+
.
.
.
.
O P O-CH2
O
O
O
N
OH
O
O P OH
O
mixture of 2’- and
3’- monophosphate
derivatives
H2O
shortened
RNA
RNA

Why does DNA contain T rather than U?
N
CH
C
O
HN
CH
C
O
C
NH2
N
N
CH
C
O
CH
cytosine uracil
H2O
Cytosine deaminates non-enzymatically to form uracil. If this
happens in DNA, it constitutes a mutation. A proof-reading
system recognize the error, and replaces the U by C.
Deamination of cytosine is of little consequence in RNA,
which is not the permanent repository of genetic information.

The phosphate groups
of DNA and RNA are
negatively charged
A phosphodiester group has a
pKa of about 1, and so will
always be ionized and negatively
charged under physiological
conditions (pH ~7).
Nucleic acids require counterions
such as Mg2+, polyamines,
histones or other proteins to
balance this charge.
5’
3’
HO-CH2
O
N
O
O P O CH2
O
O
N
O
O P O CH2
O
O
N
O
O P O CH2
O
O
O-PO3
2
N
+
M
+
M
+
M
+
M

The sugars are always in
the b-furanose (cyclic) form
5’
3’
HO-CH2
O
N
OH
O P O CH2
O
N
O
O
OH
O
O P O CH2
O
O
N
OH
O
O P O CH2
O
O
O-PO3
2
N
OH
N
3’ 1’
5’
4’
endo
C-2’ exo
The ring can adopt various puckered conformations
in which C-2’ and C-3’ are in either exo or endo
positions relative to the base and C-5’.
CH2OH
H-C-OH
H-C-OH
H-C-OH
H-C-OH
H-C=O
ribose in its
aldehyde form
b-furanose
(ring) form
HO-CH2
OH
OH
OH
O

The nucleotide base
can rotate with respect
to the sugar
The bases can adopt either
syn or anti conformations,
but anti conformations are
preferred.
syn-Adenosine
NH2
C
C N
N
N
HC
C
N
CH
O
HOCH2
OH OH
NH2
C
C
N
N
N
CH
C
N
HC
O
HOCH2
OH OH
anti-Adenosine
O
C
N
N
CH
C
CH
NH2
O
HOCH2
OH OH
syn-Cytosine anti-Cytosine
O
HOCH2
OH OH
O
N
N
C
HC
NH2
HC
C

The pattern of X-ray diffraction by DNA fibers reveals
a helical structure with steps of 3.4 and 34 Å
This x-ray diffraction by
calf thymus DNA was
measured by Franklin &
Gosling in 1952. The X
pattern is indicative of a
helix with a pitch of 34 Å
per turn. The strong
spots at the top and
bottom reveal internal
steps of 3.4 Å.
http://osulibrary.oregonstate.edu/specialcollections/coll/pauling/dna/pictures/franklin-typeBphoto.html

H-bonds between Watson-Crick base pairs stabilize a double helix
Base-pairing explains Chargaff’s rules for the base composition of DNA: A = T; G = C
A forms 2 H-bonds
with T or U
T
A
H N C-H
N
C
CH3
C
O
C
O
C
C
C
C
N
N
N
C
N
N H
H
H
H
G and C form
3 H-bonds
C
G
C-H
H
C
N
O
C
N
C
H N
H
C
C
C
C
N
N
N H
C
O
N
N H
H
H
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material” -- J.D. Watson & F.H.C. Crick, Nature 171: 737 (1953)

Base pairs fill the center of the helix; the phosphates ( ) are on the outside.
The B-form DNA helix has a diameter of about 20 Å
~20 Å
A base pair is more exposed to the solvent on one side (the “major groove”,
at the top in these views) than the other (the “minor groove”, bottom).

B-form DNA consists of a right-handed double helix with antiparallel strands
34 Å (10 bp)
per turn
major groove
minor groove
major
groove
minor
3.4 Å
per bp
These dimensions are for DNA fibers. In solution, there are ~10.5 base-pairs per turn.
5’ 3’
5’
3’

In this example, 2/4 of the Tp and 1/5 of the Gp are labeled. What would you expect if the
two strands were parallel? (Answer: 1/4 of the Tp and 2/5 of the Gp would be labeled.)
Kornberg synthesized labeled double-stranded DNA enzymatically from a-
32P-labeled 5’-nucleoside triphosphates and hydrolyzed the DNA with a
DNase that released the 3’-mononucleotides. For example, with labeled
ATP and unlabeled TTP, GTP and CTP, the sequence shown below gives:
The antiparallel orientation of the two strands was demonstrated by
synthesizing DNA enzymatically from labeled nucleoside triphosphates
DNase
... Gp + Ap + Cp + Tp + Ap + Ap + Cp + Cp + Gp ...
... Cp + Tp + Gp + Ap + Tp + Tp + Gp + Gp + Cp ...
H2O
DNA polymerase 5’ ... pGpApCpTpApApCpCpGp ... 3’
3’ ... pCpTpGpApTpTpGpGpCp ... 5’
pppA + pppT
+ pppG + pppC

NMR structure of a duplex DNA
dodecamer in the DNA-binding
site of an interferon promotor.
J. R. Huth et al., Nature Struct. Biol.
4: 657 (1997). 2eze. pdb
High-resolution structures of small DNA
molecules have been obtained by
crystallography and NMR

The two strands of the double helix
separate reversibly at high temperatures
The temperature at which this
“denaturation” or “melting”
occurs depends on the pH
and salt concentration, and
increases with the GC content
of the DNA. (The curves
drawn here are schematic.)
If the temperature is lowered,
the strands recombine. The
rate of reassociation is
inversely proportional to the
complexity of the DNA.

dsDNA
ssDNA
nucleotides
dA
dC
dG
dU
The conjugated p-electron systems of
the purine & pyrimidine bases absorb
strongly in the UV. (That’s why UV
light is mutagenic and carcinogenic.)
The absorbance of double-stranded
DNA (dsDNA) at 260 nm is less than
that of either single-stranded DNA
(ssDNA) or the free bases. This is
called “hypochromism.”
Double-stranded and single-stranded DNA differ
in their optical absorption at 260 nm

Hypochromism results from dipole-dipole interactions
between neighboring bases
The excited states of an interacting pair of molecules can be described as linear
combinations of the excited states of the individual molecules. In certain geometries,
some of the absorption strength in the near-UV moves to bands at higher energies.
individual
base
hn
individual
base
filled
orbitals
empty
orbitals
strong
absorption
band
interacting
bases
strong band near
260 nm
weaker band
near 260 nm
strong band near
260 nm
increasing
energy

Summary of the main structural features of B-form DNA
•Right-handed helix
•Two antiparallel strands held together by
Watson-Crick hydrogen bonds
•Pitch (repeat length) = 34 Å (3.4 nm)
•36o rotation between residues
•Helix diameter of 20 Å (2.0 nm)
•Wide major groove, narrow minor groove
•Chargaff’s Rules: A = T; G = C
•Charged phosphates
•Bases in anti configuration
•The strands separate at high temperatures
•The solution structure is dynamic

DNA forms other 3-dimensional
structures under some conditions
Two views of the crystal
structure of (5’-TCGCGCG-3’)2
in the DNA-binding domain of
an RNA-editing enzyme.
T. Schwartz et al. Science 284:
1841 (1999). 1qbj. pdb
Z-form DNA has a left-handed
helix with 12 bp per turn, ~18 Å
diameter, and alternating Syn
and anti conformations. It
probably occurs in vivo only in
short stretches.

Hoogsteen pairs stabilize triplex DNA structures
A protonated cytidine can form two H-
bonds to the guanosine of a G-C pair.
protonated C
C-H
H
C
N
O
C
N
C
H N
H
C
G
C
C
C
C
N
N
N H
C
O
N
N H
H
H
N
C
C
N
C
O
N
C
H
H
H
H
H
+
A thymidine can form two H-bonds
to the adenosine of an A-T pair.
T
A
H N C-H
N
C
CH3
C
O
C
O
C
C
C
C
N
N
N
C
N
N H
H
H
H
N C
H
N
C CH3
C
O
C
O
H
T

xx
Guanine
(keto form)
H2N-C
C
C
HN
N
N
CH
C
O
N
Thymine
(keto form)
N
C
C
O
HN
CH
C
O
CH3
N
N
C
C
OH
CH
C
O
CH3
Thymine
(enol form)
N
OH
H2N-C
C
C
N
N
CH
C
N
Guanine
(enol form)
The ring NH atoms
of G and T have
pKa values of
about 9. At
physiological pH,
about 99% of the
base is in the keto
form and 1% in the
enol form.
Tautomeric forms of G & T can cause mutations
due to mispairing during DNA replication

Palindromic* sequences (inverted repeats) in DNA
or RNA can form hairpin or cruciform structures
inverted repeats in an antiparallel double helix
3’
5’
5’
3’
T G C G A T A C T C A T C G C A
A C G C T A T G A G T A G C G T
hairpin
C
A C
T
3’
5’
T
A
G
C
G
T
A
T
C
G
C
A
T
G A
G
C
A C
T
cruciform
3’
5’
5’
3’
T
A
G
C
G
T
A
T
C
G
C
A
T
G
C
G
A
T
A
C
G
C
T
A
Mirror repeats cannot form these structures.
*A palindrome reads the same in either direction (“Radar,” “Madam, I’m Adam”).

Cells contain a variety of types of RNA
with different functions
Principle kinds of RNA in E. coli
Type Sed. Coef. Mol. Wt. Residues % of total RNA
mRNA 6 - 25 25,000 - 1,000,000 75 -3000 ~2
tRNA ~4 23,000 - 30,000 73 - 94 16
rRNA 5 35,000 120
16 550,000 1,542 82
23 1,100,000 2,904
Eucaryotic cells contain an additional type, small nuclear RNA (snRNA).

Most RNA molecules consist of a single strand that
folds back on itself to form double-helical regions
In RNA, G pairs with C
and A pairs with U.
The loops and
hairpins have few
or no base-pairs
single
strands
bulge
internal
loop
hairpin
A-form
double
helix
CGU
GCA
A

licorice
representation
The 3-dimensional structures of even relatively
small RNA molecules can be very complex
5’
3’
tube
representation
Crystal
structure of
t-RNAphe
4nta.pdb

Structure of a “hammerhead” ribozyme
Ribozymes are RNA molecules
that catalyze events in RNA
processing. Hammerhead
ribozymes are small viral RNAs
with two chains.
chains A & B
1mme.pdb
chain A

Single-nucleotide
polymorphisms can
change the
secondary structure
of mRNA
These are predicted structures and
calculated free energies of folding (probably
not very accurate) for part of the mRNA for
catechol O-methyltransferase. The “LPS”
haplotype results in about 25-times as much
protein synthesis as “HPS.”

DNA base sequences can be determined by using
DNA polymerase and dideoxynucleotides
Synthesize DNA enzymatically in the presence of a small amount of a
dideoxynucleotide (e.g., ddTTP) and larger amounts of ordinary ATP, GTP,
CTP and TTP, using a fluorescently tagged primer and one strand of the DNA
to be sequenced as a template. DNA polymerase synthesizes the new strand
in the 5’  3’ direction. The dideoxynucleotide interrupts DNA synthesis when
it is incorporated. This happens at random points where the corresponding
base occurs in the sequence. In this illustration, each DNA fragment ends in
ddT and is labeled with a red dye.
O
O P O-CH2
O
O
T
O P
O
O
O P
O
O
ddTTP
synthesis
C G G C G T A C A G C A T T G A A
G C C G C A T G T C G T A A C T T
5’ primer 3’
template
3’ 5’ (continued …)

DNA sequencing can be automated
Repeat the procedure with
ddC, ddA and ddG, using a
different fluorescent dye to
tag the primer in each case.
Then mix all the DNA
fragments and separate them
according to size by
electrophoresis.
Separating the DNA
fragments gives a ladder of
bands, each ending in a
particular dideoxynucleotide
at its 3’ end and tagged at the
5’ end with the corresponding
dye. This procedure has
been automated in machines
that can sequence more than
3x106 bases in a month.
synthesis
5’ primer 3’
template
3’ 5’
G C T C G A T A
C G G A A C T G A
C G A G C T A T
G C C T T G A C T
C G A G C T A T
C A
G C T T G C T

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