Nucleotides and Nucleic Acids
The Basics
Deoxyribonucleic Acid (DNA) and
Ribonucleic Acid (RNA)
Nucleic acids were first
isolated by Friedrich
Miescher in 1869.
The monomers constituting
nucleic acid polymers are
nucleotides:
Purine and Pyrimidine Bases in DNA and
RNA
Deoxyribonucleotides
Ribonucleotides
The Bases Absorb UV Light
DNA is the Genetic Material
“Transforming
principle” of
pneumonia-causing
bacteria (Griffith, 1928)
DNA is the
“transforming principle”
(Avery, McLeod and
McCarty,
1944)
“Waring
Blender
Experiment”
(Hershey
and Chase,
1952)
Avery, McLeod
and McCarty,
1944
“Waring
Blender
Experiment”
(Hershey and
Chase,
1952)
Three-Dimensional Structure of
Deoxyribonucleic Acid (DNA)
Double Helix: 50 Years of DNA
http://www.nature.com/nature/dna50/
An Early Conjecture about DNA Structure
Tetranucleotide
hypothesis
(Levene, 1910)
X-Ray Diffraction Pattern of DNA Fibers
Franklin and Wilkins, 1953
Rosalind Franklin
Maurice Wilkins
X-Ray Diffraction Pattern of DNA Fibers
Franklin and Wilkins, 1953
Incorrect Triple-Helical Structure
Pauling and Corey, 1953
Chargaff’s Rules:
A=T and G=C
purines = pyrimidines
Chargaff’s Rules
Tautomerism of the Bases
April 25, 1953
MOLECULAR STRUCTURE OF NUCLEIC ACIDS
A Structure for Deoxyribose Nucleic Acid
We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.).
This structure has novel features which are of considerable biological
interest.
…
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
Medical Research Council Unit for the Study of Molecular Structure of Biological
Systems, Cavendish Laboratory, Cambridge.
Watson-Crick Base Pairs
Elements of Structure in DNA Double
Helix
The DNA Double Helix
Forms of DNA
The A, B and Z Forms of DNA
•Most DNA in the cell is in the B form.
•A small amount of the DNA in the cell may
locally adopt the Z conformation (where
sequence has alternating purine and
pyrimidine bases).
•The A form of DNA is found in dehydrated
samples of DNA but not normally in the cell.
However, double-stranded RNA and DNARNA hybrids form helices resembling the A
form of DNA.
Z-DNA: Left-Handed Helix
Alexander Rich, 1979
Proteins that Bind Z-DNA
X-Ray structure of two ADAR1 Z domains in complex with
Z-DNA.
Nucleotide Conformation
Sugar-phosphate backbone is conformationally
constrained (O4’ generally gauche to O5’ since torsion
angle about the C4’-C5’ bond is restricted).
Sugar Conformation
In A-DNA: sugar pucker is C3’-endo.
In B-DNA: C2’-endo.
In Z-DNA: pyrimidines are C2’-endo, but purines are C3’-endo.
Orientation of Bases with Respect to
Sugar
In A- and B-DNA, all bases are in anti orientation.
In Z-DNA, pyrimidines are anti, but purines are syn.
Local Deviations from the Idealized B-DNA
Structure
Properties of Naturally Some Occurring
DNA Molecules
Haploid human genome: 23 chromosomes of ~5 × 107 bp to
~25 × 107 bp each for a total of ~3 × 109 bp.
Chargaff’s Rules Redux
DNA Replication and Transcription
into RNA
Genetic Information Flow:
DNA
RNA
Protein
“The Central Dogma”
Francis Crick, 1957
Replication, Transcription and Translation
Replication and
transcription
occur in the nucleus
Translation occurs
in the cytoplasm
Polynucleotide Synthesis
DNA Replication
DNA Replication
Hydrolysis of Pyrophosphate
DGo’ = -33.5 kJ/mol
Three Possible Models of DNA Replication
Meselson-Stahl Experiment:
Semiconservative DNA Replication
Gene Expression (Transcription, Splicing and
Translation)
Expression of a hypothetical eukaryotic gene
DNA Transcription into RNA
Regulation of Gene Expression
Catabolite activator protein (CAP)-cAMP, a bacterial
transcription factor, bound to DNA
Example of DNA Sequence Recognition
by a DNA-Binding Protein
434 phage repressor protein
mRNA Splicing
RNA Translation into Protein
The Genetic Code
Ribosomal Peptidyl Transferase Activity
Note: the catalytic component of the ribosome’s peptidyl transferase activity
is RNA; it’s an example of a catalytic RNA or ribozyme.
Stability of Nucleic Acids
The DNA Duplex Can Be Reversibly
Denatured (Melted)
Factors Determining DNA Duplex Stability
In order for the double helix to form under given conditions, forces favoring duplex
formation must outweigh disfavoring factors, so that the ∆G for duplex formation under
those conditions is negative.
Factors Stabilizing the DNA Duplex
1. “Hydrophobic interactions,” base stacking (vertical base
stacking interactions make duplex formation enthalpically
favored, although entropically opposed, unlike the Hydrophobic
Effect involved in protein folding and lipid bilayer formation)
2. Ionic interactions (duplex becomes more stable as ionic
strength increases, since presence of positive counterions
partially neutralizes negative charges of backbone phosphates)
3. Hydrogen bonding between base pairs
Under conditions in which the duplex forms spontaneously, stabilizing forces outweigh
destabilizing forces, so that ∆G of duplex formation is negative.
London Dispersion Forces in Base Stacking
(Pi-Pi Stacking)
Factors Destabilizing the DNA Duplex
1. Higher entropy of denatured random coil (denaturation is
temperature-dependent since relevant term is -T∆S in the
Gibb’s free energy equation, ∆G = ∆H - T∆S)
2. Charge-charge repulsion of backbone phosphates (so
absence of positive counterion would favor denaturation)
Under denaturing conditions, destabilizing factors dominate over stabilizing, so that
∆G of denaturation becomes negative (and ∆G of duplex formation now has a
positive sign).
Denaturation of DNA Duplex
Increasing T
makes
denaturation
favorable
Denaturation and
renaturation
of DNA duplex are
cooperative
processes.
Hyperchromic Shift
Stability of Double Helix and Melting
Temperature Increase as GC Content
Increases
Melting Temperature (Tm):
∆G (at Tm) = ∆H - Tm∆S = 0
So: Tm = ∆H/∆S
GC-rich duplex is enthalpically
more stable than AT-rich:
While ∆S of melting per bp is about
the same for all DNA molecules,
∆H (melting GC-rich DNA) >
∆H (melting AT-rich DNA)
Instability Due to Reactivity:
Individual RNA Strands Chemically Less
Stable than DNA
2’-Hydroxyl
participates in basecatalyzed
intramolecular
nucleophilic attack on
phosphorus, breaking
the phosphodiester
backbone and
forming a 2’,3’ cyclic
phosphate, with
further hydrolysis to
2’- and 3’monophosphate
products.
Thymine in Place of Uracil Makes DNA
More Genetically Stable
Spontaneous
deamination
= mutagenic base
substitution
Base pairs with guanine
A DNA repair enzyme called uracil-DNA
glycosylase removes uracil in DNA and
replaces it with cytosine, undoing the mutagenic
damage.
(Normal thymine in DNA is not removed.)
Base pairs with adenine
Other Nucleic Acid Structures
Non-Watson-Crick Base Pairing,
e.g., Hoogsteen Base Pairing
Triple Helical DNA: H-DNA
H-DNA structure can form when
you have a homopurine stretch
on a strand (so homopyrimidine
stretch on the other strand).
Self-Complementary Nucleic Acid Strands
and Hairpins
Transfer RNA (tRNA) Structure
Palindromic DNA Sequences:
Potential to Form Cruciform Structures (Double Hairpins)
Some Palindromes:
Madam, I’m Adam
And E.T. saw waste DNA.
No, Mel Gibson is a casino's big lemon.
A man, a plan, a canal: Panama!
Palindromes and Restriction Endonucleases
Another reason palindromes are important:
Type II restriction enzymes are site-specific endonucleases used in molecular biology research (such as gene cloning)
that recognize specific palindromic DNA sequences.
X-ray crystal
structure of
Eco RI bound
to DNA
DNA cleavage products:
Sticky ends (e.g., Eco RI):
5’-G-3’
5’-AATTC-3’
3’-CTTAA-5’
3’-G-5’
Blunt ends (e.g., Sma I):
5’-CCC-3’
5’-GGG-3’
3’-GGG-5’
3’-CCC-5’
Sanger Dideoxy Method of DNA
Sequencing
The most commonly used of two methods for DNA
sequencing. The other method, not as used today, is the
Maxam-Gilbert chemical cleavage method.
NTP, dNTP and ddNTP
(Dideoxynucleoside Triphosphate)
Structures
Sanger Dideoxy Method of DNA Sequencing
Fred Sanger
Automated DNA Sequencing
DNA Topology and Supercoiling
Relaxed vs. Supercoiled DNA
Positive and Negative Supercoiling
positive supercoil =
left-handed =
overwound DNA
negative supercoil =
right-handed =
underwound DNA
L=T+W
• L or Lk = linking number (number of times one strand
crosses the other)
• T = twist (number of helical turns; for B-DNA, T = #
bp divided by ~10.5 bp/turn)
• W = writhe (number of supercoils)
Superhelical Density: s = DL/L0 = W/T
(L0 = linking number of relaxed molecule = T,
since W = 0 in relaxed molecule)
Formation of DNA Supercoil
Supercoiling of DNA Shown by Gel
Electrophoresis
Agorose Gel Stained with Ethidium Bromide
Supercoiled circular DNA migrates faster
during agarose gel electrophoresis than
does relaxed circular DNA (or linear DNA).
Ethidium = fluorescent
intercalating agent used to
stain DNA in agarose gels
Type I Topoisomerases
Ο
Ο
Ο
Ο
•∆L = ±1 per cycle
•Cleaves a single
strand
•Passes broken single
strand around the other,
then rejoins strands
•Does not require ATP
•Relaxes supercoiled
DNA
Structure of a Type I Topoisomerase
Type II Topoisomerases
•∆L = ±2 per cycle
•Cleaves both strands
•Passes unbroken part of
duplex through doublestrand break, then rejoins
strands
•Requires ATP
•Relaxes supercoiled DNA
•Some type II enzymes (like
DNA gyrase) can add
negative supercoils
Topological Interconversions Catalyzed
by Type II Topoisomerase
Relaxation
Catenation and
Decatenation
Knotting and
Unknotting
X-Ray Crystal Structure of a Type II
Topoisomerase
Chromosomes and Chromatin: HigherOrder DNA Structure
The Nucleosome DNA (146 bp) wrapped around
octamer of core histone proteins (+
linker DNA = ~200 bp)
Negative Supercoiling of DNA is
Important Biologically
•
Negatively supercoiled DNA is equivalent to underwound DNA.
This makes it easier to separate strands during replication and
transcription.
•
In eukaryotes, formation of nucleosomes results in torsional strain in
the DNA molecule (equivalent to ~1.5-1.8 supercoils/nucleosome
particle theoretically; actual value is ~1), which is relieved by
topoisomerases. This results in DNA that is negatively supercoiled
once histone proteins are removed.
•
In prokaryotes, an enzyme called DNA gyrase (a type II
topoisomerase) generates negative supercoils.