Nucleic Acids
CH339K
Monomers: Nucleotides
Component 1: 5-Carbon Sugar
Ribose vs. Deoxyribose
• Difference in components of DNA and RNA
• Extra hydroxyl makes RNAs much more reactive
Sugar Pucker
• Furanoses are not planar
• Can pucker out of the plane of the ring at C2 or C3
• Pucker effects higher order structures (or vice-versa)
Component 2: Nitrogenous Base
Purines
Pyrimidines
• Cytosine and Thymine in DNA
• Cytosine and Uracil in RNA
Component 3: Phosphate
Nomenclature
Names of Base Derivatives
Base
Nucleoside
5'-Nucleotide
Adenine
(Deoxy)Adenosine
(Deoxy)Adenosine-5'-monophosphate
Guanine
(Deoxy)Guanosine
(Deoxy)Guanosine-5'-monophosphate
Cytosine
(Deoxy)Cytidine
(Deoxy)Cytidine-5'-monophosphate
Thymine
(Deoxy)Thymidine
(Deoxy)Thymidine-5'-monophosphate
Uracil
(Deoxy)Uridine
(Deoxy)Uridine-5’-monophosphate
Syn and Anti Conformations
Syn / anti energetics
From: Neidle, S. (2008) Principle of Nucleic Acid
Structure Elsevier, London, pg. 33
Condensation – Polymer Formation
Phosphodiester Linkages
Simple Condensation is Energetically
Unfavorable
DGo‘≈ +25 kJ/mol
Keq=4.15*10-5
Synthesis is from the triphosphate
Energetics:
Phosphodiester formation - +25 kJ/mol
nTP cleavage –
-31 kJ/mol
Pyrophosphate cleavage - -19 kJ/mol
Keq = 24100
Tautomeric Forms of Bases
-NHx groups can be in the amino or imino
conformation
=O groups can be in the keto or enol
conformation
The predominant form for the free base is
not necessarily the predominant form in the
nucleotide
Lack of basic O-Chem knowledge caused
problems for Watson and Crick when they
were trying to figure out the structure of
DNA
Keto
Enol
Base Pairing
keto
amino
Cytosine
amino
keto
Guanine
Animation
Secondary Structure of Nucleic Acids
• Helical
• Result of base
pairing
• Defined by
– Pitch
– Rise
– In turn governed
by structure of the
monomers
B Helix
B form
Helical Sense
Diameter
Right handed
~20Å
Base pairs per helical
turn
10
Helical twist per base
pair
36°
Helix pitch (rise per
turn)
34 Å
Helix rise per base pair
3.4 Å
Base tilt normal to the
axis
6°
Major groove
Wide & deep
Minor groove
Narrow &
deep
Sugar pucker
C2'-endo
Glycosidic bond
Anti
Typical DNA
Determination of helix parameters
Rosalind Franklin’s Diffraction Photo of B-DNA
A Helix
A form
Helical Sense
Diameter
Right handed
~26 Å
Base pairs per helical turn
11
Helical twist per base pair
33°
Helix pitch (rise per turn)
28 Å
Helix rise per base pair
2.6 Å
Base tilt normal to the
axis
20°
Major groove
Narrow & deep
Minor groove
Wide & shallow
Sugar pucker
C3'-endo
Glycosidic bond
Anti
RNA, DNA/RNA hybrids,
dehydrated DNA
Z Helix
Z form
Helical Sense
Diameter
Left handed
~18 Å
Base pairs per helical
turn
12 (6 dimers)
Helical twist per base
pair
60° (per dimer)
Helix pitch (rise per
turn)
45 Å
Helix rise per base pair
3.7 Å
Base tilt normal to the
axis
7°
Major groove
Flat
Minor groove
Narrow & deep
Sugar pucker
C2'-endo (pyrimidines)
C3'-endo (purines)
Glycosidic bond
Anti (pyrimidines)
Syn (purines)
Alternating Purine-Pyrimidine
Z DNA Function?
• Z DNA is antigenic
• Antibodies are found in autoimmune
disorders like systemic lupus erythematosus
• Antibodies bind to puffs in Drosophila
polytene chromosomes
• Also bind macronuclei of ciliates
• Z DNA-prone sequences found in
transcription start sites
• May act as spacer between RNA
polymerases
• Z DNA binding proteins required for
pathogenicity by vaccinia and smallpox
Helix Parameters Summarized
A form
Helical Sense
B form
Z form
Right handed
Right handed
Left handed
~26 Å
~20Å
~18 Å
Base pairs per helical turn
11
10
12 (6 dimers)
Helical twist per base pair
33°
36°
60° (per dimer)
Helix pitch (rise per turn)
28 Å
34 Å
45 Å
Helix rise per base pair
2.6 Å
3.4 Å
3.7 Å
20°
6°
7°
Major groove
Narrow & deep
Wide & deep
Flat
Minor groove
Wide & shallow
Narrow & deep
Narrow & deep
C2'-endo
C2'-endo (pyrimidines)
C3'-endo (purines)
Anti
Anti (pyrimidines)
Syn (purines)
Diameter
Base tilt normal to the
axis
Sugar pucker
Glycosidic bond
C3'-endo
Anti
Major and Minor Grooves
Grooves provide access
to base sequence
• Telomere binding
protein
• a-helix fits into major
groove
• Side chains can
recognize bases
Another Example
cro Repressor protein of
bacteriophage l.
Small (66 amino acids)
Forms dimers
Binds to specific sites on DNA that
activate / deactivate genes
Expression of cro results in the phage
entering the lytic cycle
Absorption of UV Light
UV Absorption Spectrophotometry
Beer-Lambert Law
I
T ransmitta
nce : T   10cl
Io
or alternatively
Absorbance: A   logT  cl
c = concentration
l = path length
 = extinction coefficient
An Absorbance = 2 means that only 1% of the incident beam is getting through.
Transmittance and
Absorbance
Absorbance vs. Concentration
Transmittance vs. Concentration
Physical Properties - Absorbance
Physical Properties - Hypochromicity
• Stacked bases in nucleic acids absorb less
ultraviolet light than do unstacked bases, an
effect called hypochromism
• Rules of thumb:
– 280 dsDNA: 20
– 280 ssDNA/RNA: 37.5
– 280 small oligonucleotides: 50
1) Calculated spectrum of equivalent mixture of free nucleotides
2) Double stranded RNA (38% G+C)
3) Single stranded RNA (38% G+C)
From Cox, R. A. (1970) Conformation of Nucleic Acids and the Analysis of
the Hypochromic Effect, Biochem. J. (1970) 120, 539-547
Denaturation: “Melting”
• Heat, alkali cause the
double helix to unwind
• As H-bonds break, they
form “bubbles” in the
helix
• As the equilibrium shifts
towards H-bonds
breaking, the bubbles
coalesce
• The strands come apart
As temperature increases, local denatured
regions coalesce
Effect of G+C content on Tm
ss Bubbles Coalesce until Strands
Separate
Effects of changing DHo’ and DSo’
Artificially generated curves
1.0
0.9
0.7
Decrease
DHo' by 5%
0.6
Decrease
DSo' by 5%
0.5
0.4
0.3
0.2
0.1
T (K)
425
400
375
350
325
300
0.0
275
Fraction Single Stranded
0.8
DNA Sequencing – Sanger Method
DNA Sequencing - Sequencers
Polymerase Chain Reaction
(aka DNA Amplification)
Internal Structure
Palindromes and inverted
repeats tend to be sites for
recognition by proteins
Palindromes:
Kay, a red nude, peeped under a yak
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Internal Structure (cont.)
Replication Origin of Duck Hepatitis B
Nonstandard Base Pairs
Triplex DNA Structure
A) Duplex DNA Structure
B) Triplex DNA with 3rd Strand in Major Groove
Bissler, John J. (2007) Triplex DNA and human disease, Front. Biosci. 12: 4536-4546.
Duplex, Triplex, and Quadraplex
Quadraplexes are found in telomeres
Telomeres contain repeats of d(GGTTAG), which form quadraplexes.
Nucleic acids can
form higher – order
three dimensional
structures…
…and it’s a good thing.
Tertiary Structures - tRNAs
tRNAs can
contain a
variety of
modified
nucleotide
bases
Tertiary Structure - Viroids
• Viroids are small, naked circular, mostly
double-stranded RNAs which infect plants
• Host RNA Polymerase copies the RNA many
times
• Self-cleavage into individual lengths
• Host ligases close into circles
Potato
Spindle
Tuber
Viroid
African oil palm with
cadang-cadang like
viroid disease
Frequency of Cadang-Cadang in Coconut palms from two
Phillippine provinces 1951-1976
From Zelazny, B., and Pacumbaba, E. (1982) Plant Disease 66: 547549.
Tertiary Structures (cont.)
Examples of some
specialized RNAs
E. coli 16S ribosomal RNA
Nucleases
• Nucleic acids can be hydrolyzed enzymatically by
nucleases;
• Nucleases belong to the class of
phosphodiesterases;
– Cleavage at the 3’ side by “a” type nucleases
(leaves 5’ phosphate);
– Cleavage at the 5’ side by “b” type nucleases
(leaves 3’ phosphate);
– Endonucleases cleave in the middle of the NA;
– Exonucleases cleave from the ends.
• DNases act on DNA; RNases act on RNA.
Examples
b-type endo
a-type 3’ exo
5’ p-A-p-G-p-G-p-T-p-C-p-C-p-T-p-A-OH 3’
b-type 5’ exo
Word of the Day: Processivity - The ability of an enzyme to
repetitively continue its catalytic function without
dissociating from its substrate.
(The exonuclease examples above are not processive)
Examples
Enzyme
Substrate
Type
Pancreatic RNase
RNA
b-type (5’) endo
Snake Venom Phosphodiesterase
RNA / DNA
a-type (3’) exo
Spleen Nuclease
RNA / DNA
b-type (5’)exo
Examples
From Smith, C., and Wood, E. J. (1991) Biological Molecules, Springer, New York, pg 188
Restriction Systems - Bacteriophage
Bacteriophage T4
Restriction Systems - Bacteriophage
Restriction
Endonucleases
• Phage hatched from the
R strain reinfect the R
strain easily.
• Phage hatched from the
K strain reinfect the K
strain easily.
• Phage from the R strain
are restricted on K
• Phage from the K strain
are restricted on R.
Infectivity
~1 x 10-4
Infectivity
~1
E. coli R
Kablooey!
Infectivity
~1
Kablooey!
E. coli K
Infectivity
~1 x 10-4
Restriction due to endonuclease /
methylase system
The endonuclease and the methylase recognize the same sequence
The endonuclease will not cut the methylated DNA
Host can discriminate its own DNA from that of a virus if the virus is raised in a
bug with a different restriction system
Protective Role of Restriction Systems
Example of a restriction modification system
EcoR1 (first restriction system from E. coli strain R) recognizes a 6-base
palindrome:
5'-GAATTC-3'
3'-CTTAAG-5'
The methylase puts a methyl group on the underlined adenosines if the
sequence is not methylated. The nuclease clips each strand
between the 5' G and A of the unmethylated recognition site
5'-G
AATTC-3'
3'-CTTAA
G-5'
The resulting overhangs are "sticky ends" - they will base pair with a
complementary sequence.
Cloning using Restriction
Endonucleases
There are a zillion REs for just about
any palindrome
(Enzymes for 234 recognition sites available from New
England Biolabs as of March 2010)
DNA Modifying Agents as Drugs
Intercalating Agents
• Stack between base
pairs
• Ethidium is used as a
fluorescent DNA stain
• Acridine is also used
as a stain for DNA
(green) and RNA (red)
• Actinomycin inhibits
transcription by binding
at the start site
Ethidium Bromide Intercalated into DNA
DNA stained with ethidium bromide
Reactivity of Ribonucleic Acid Due to
the 2’-Hydroxyl
Base 1
Base 1
Base 1
O
O
O
O
H
H
H
O
H
OH
O P O-
base
Base 2
H
H
H
O
O
H
H
O
H
OH
O P O-O
H
H
O
H
O H2O
O
O
O
H
H
H
H
O
H
OH
O P O-O
H
H
P
Base 2
O P O-
O
O
H
O
O
O
O
Base 1
O-
H
H
H
O
OH
O P OOBase 2
Base 2
HO
HO
O
O
H
H
H
O
H
OH
O P O-O
H
H
H
O
H
OH
O P O-O
Supercoiling
• DNA in “relaxed” state - 10.4 bp/turn
• If DNA is twisted, the strands become more tightly or
more loosely wound: supercoiling
– in direction of helix = “positive supercoiling”
– in the opposite direction = “negative supercoiling”
• In nature, most DNA has a slight negative
supercoiling that is introduced by enzymes called
topoisomerases (counteract effect of transcription
and replication)
Supercoiling
•
•
•
•
•
Can open helix
Overwind or underwind
Changes Linking Nbr
L=T+W
Underwound DNA is
primed to uncoil
–
–
–
–
Transcription
Replication
Recombination
Z-DNA formation
Linking Numbers
Negative and Positive Supercoils
Supercoiling in a viral DNA
Different levels of supercoiling in Simian Virus 40 (SV40) DNA..
SV40 may (or may not) be involved in causing human tumors.
Those of us inoculated for polio prior to 1962 were probably
exposed to SV40 as a contaminant of the polio vaccine.
Supercoiling of Constrained Linear
DNA
Supercoiling: Energetic Considerations
•
Because there are ~10.4 bp/turn in B-helical DNA, the
relaxed Linking Number is
Lo = bp / 10.4
•
Upon supercoiling, the change in L is
ΔL = L − Lo
•
We can define superhelical density as
σ = ΔL / Lo
•
The free energy involved in supercoiling is related to s
ΔG / N = KRTσ2 (usually shown as ΔG = KNRTσ2)
where N = number of (constrained) base pairs and
K depends on the solution (ionic strength, concentration, etc.)
Supercoiling (cont.)
• Wrapping of DNA around nucleosome
requires s ≈ -0.05
Type 1
Topoisomerases
Cut 1 strand of the DNA
Change L by 1
Involved in protein
synthesis control
Type 2 Topoisomerases
Cut both strands simultaneously
Change L by 2
Most well known is DNA Gyrase
In presence of ATP can induce
supercoiling
Unwinds DNA ahead of
replication fork
Topoisomerase Action
Simian virus 40 (SV40) DNA incubated w/ a human topoisomerase for 0, 1,
3, 6, 10, and 30 minutes, going from an average of 25 superhelical
turns to 0 (relaxed)
Riddle me this, Doc…
…why does DNA use Thymine instead of
uracil? Seems like a waste of complexity.
Spontaneous Deamination
Asn Pro Gly Cys
AAT CCT GGC TGT
TTA GGA CCG ACA
Asn Ser Gly Cys
AAT TCT GGC TGT
TTA AGA CCG ACA
Frequency: 100 – 500 times per human cell per day.
That’s about 1 - 5 * 1015 per person per day.
DNA Mismatch Repair
DNA has a system to
recognize uradines in the
DNA strand
Glycosylase clips of the
uracil base
An endonuclease clips out
the sugar phosphate
Polymerase fills the gap
Expectations
• Know structures of nucleotides and components.
• Understand the linear, directional, backbone / base
structure of the polymer.
• Understand base pairing.
• Understand the properties of the helix types and what
types of nucleic acids assume which forms.
• Beer’s law
• Difference between primary, secondary, and tertiary
structure.
• Different types of nucleases; what are restriction
systems?
• Meaning and significance of supercoiling;
topoisomerases.
• What the heck is spontaneous deamination?