Module 7: Nucleic Acids

Learning Objectives

1a. Draw and name purine/pyrimidine nucleobases, nucleosides and

nucleotides.
1b. Describe base pairing in nucleic acids.
2. Describe structural organization in the DNA double helix and the
cellular functions of DNA and RNA.

Pre-Class Assignment: Memorize purine/pyrimidines: 07.nucleotides.pdf

Map the factors that create hierarchical structure in nucleic acids:
1° nucleobases, phosphodiester backbone; titration, mutation.
2° base pairing; double helix. 4° nucleosomes.

Reference: Lehninger Ch. 8

http://en.wikipedia.org/wiki/Dna http://en.wikipedia.org/wiki/Base_pair
http://en.wikipedia.org/wiki/Double_helix
© R. Hills
• Cell division • Gene expression (mRNA)
mRNA: messenger RNA
Gets transported from the
nucleus to the cytoplasm for
protein synthesis.
 Gene Expression

• A promoter is a region of DNA that initiates transcription of a particular gene. Promoters

are located near the transcription start sites of genes, upstream on the same DNA strand.
Promoters are a non-coding DNA sequence, or regulatory element, 100–1000 base pairs
long. The core promoter region contains a TATA box, with A:T base pairs that are easily
unwound.
• A nuclear transcription factor is a DNA binding protein that controls the rate of
transcription of genetic information from DNA to messenger RNA, by binding to a specific
DNA sequence. The function of TFs is to regulate—turn on and off—genes in order to
make sure that they are expressed in the right cell at the right time and in the right
amount throughout the life of the cell and the organism.
• Groups of TFs function in a coordinated fashion to direct cell division, cell growth, and
cell death throughout life; cell migration and organization (body plan) during embryonic
development; and intermittently in response to signals from outside the cell, such as a
hormone. There are up to 2600 TFs in the human genome.
• TFs work alone or with other proteins in a complex, by promoting (as an activator), or
blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs
the transcription of genetic information from DNA to RNA) to specific genes.
• Steroids are hormones in the body that bind and activate nuclear transcription factors.
 Nitrogen Bases
DNA (deoxyribonucleic acid and RNA (ribonucleic acid) are
polynucleotides. Their building blocks are nucleotides, which are phosphate
esters of nucleosides. Nucleosides are glycosides of β-ribofuranose and a
nucleobase. Nucleobases are nitrogenous bases derived from purine or
pyrimidine:
pyrimidine
What are the lone pair orbitals?

purine

• The low pKa=1.2 for protonated

pyrimidine (compare to pyridine,
module 2) is due to the less electron
rich ring of the pi bonded nitrogens.

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 Nucleobases are
neutral at pH 7
(uncharged in DNA)

• pKa’s of derived
compounds are shifted
from their parent
compound due to electron
withdrawing groups.

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 DNA vs. RNA function
• DNA is the central repository of genetic information.

• If DNA is damaged beyond repair it triggers programmed cell death

(apoptosis), but if this pathway is damaged -> cancer.

• RNA helps in the expression of this information, as an intermediary

between DNA and the synthesis of proteins, and it has several other
important roles in the cell as well.

• Genes are segments of DNA that specify particular proteins or particular

species of RNA that are used in structural or catalytic roles by the cell. A
chromosome is a single DNA molecule carrying a set of genes.

• A genome is the complete genetic complement of a cell. A simple

organism such as the bacterium Escherichia coli might have a genome of
4,000 genes; humans are thought to have about 30,000 distinct genes.

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 DNA vs. RNA primary structure

• “Primary structure” = covalent structure and sequence.

• Think of it like a protein.

– DNA and RNA have a Sidechain

sugar-phosphate backbone.

– DNA and RNA have

sidechains, which are
heteroaromatic bases
that can hydrogen bond
with each other: edge-
to-edge base pairing.

9 Sugar-phosphate backbone
 • Pentose (i.e. 5-carbon) sugar in a furanose form (i.e. 5-
membered ring consisting of 4 carbons and a cyclic ether
oxygen).

• The type of pentose defines whether it’s DNA or RNA:

– 2-deoxyribose: DNA = deoxyribonucleic acid
– ribose: RNA = ribonucleic acid
– only difference is at C2’:

DNA: X = -H
RNA: X = -OH
X

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 • Phosphate acts as a
bridging link
(phosphodiester) between
furanoses.
• Negative (−1) charge
at pH 7.

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 “Sidechains” of Nucleic Acids:
nitrogenous bases

• Called bases because they are weakly basic (poor bases

with pKa < 7).
• 5 major types (compare to the 20 amino acids).
• 3 pyrimidines:
– cytosine (C), thymine (T), uracil (U)
– One 6-membered ring.
– T appears in DNA
– U appears in RNA
• 2 purines:
– adenine (A) and guanine (G)
– Two rings: 6-membered ring fused to a 5-membered ring.

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• Sugar atom numbers are denoted
with a prime (‘) to distinguish
them from purine/pyrimidine atom
numbers.

= site of attachment to
furanose C1’ (glycosidic
bond)

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 • The N-glycosidic bond
torsion angle is usually in
the anti configuration (180°)
rather than syn (0°).

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 DNA Base Pairing
• In Watson-Crick base pairing,
adenine (A) hydrogen bonds with
thymine (T) and guanine (G) hydrogen
bonds with cytosine (C) as shown.

Planar sp2
hydrogen bonding

• In contrast, non-Watson-Crick, or
wobble, base pairs involve complex
structures (e.g. A+ŸC). Non-canonical
pairing is rare but can lead to point
mutations.

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 Nucleosides: pentose-1’-base
• Pyrimidine nucleoside names end in “-dine” (cytidine, thymidine, uridine)
• Purine nucleoside names end in “-sine” (adenosine, guanosine)

Nucleotides: 5’-phosphate-pentose-1’-base
• A string of covalently linked nucleotides make a single stranded DNA or RNA
molecule (polynucleotide)
– Pyrimidine nucleotides end in “-dylate” (cytidylic acid: cytidylate; thymidylic
acid: thymidylate; uridylic acid: uridylate)
– Purine nucleotides end in “-nylate” (adenylic acid: adenylate; guanylic acid:
guanylate)
• Have 1, 2, or 3 phosphates (usually at C5’)
– For clarity, can be named as nucleoside + phosphate
• adenosine monophosphate (AMP)
• adenosine diphosphate (ADP)
• adenosine triphosphate (ATP)
• High energy bonds: phosphoanhydride hydrolysis yields 30 kJ/mol (14 kJ for
phosphoesters) standard free energy (even more in physiologic conditions) that can be
used to drive non-spontaneous cellular processes

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Structure of nucleotides 14 kJ

30 kJ
30 kJ

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 Nucleotides: Other functions

• Enzyme cofactors, or coenzyme (nicotinamide adenine dinucleotide)

• Signaling: cyclic AMP

• Adenylate cyclase catalyzes cAMP synthesis: ATP -> cAMP + PPi

(pyrophosphate – spontaneously hydrolyzes to inorganic phosphate, Pi).
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 Sequence Naming Convention
• Polynucleotide sequences are named from the 5’ end to the 3’ end. DNA
sequence TGGAC implies 5’-TGGAC-3’.
• The 5’ end has a phosphate group attached (nucleotide = nucleoside 5’-
monophosphate); 3’ end just has a hydroxyl.

• DNA/RNA polymerases extend

a growing nucleotide chain by
incorporating a free
(complementary) nucleoside
triphosphate at the 3' end of a
short starter (primer) sequence.
• Energy released from
phosphoanhydride hydrolysis
goes into the new phosphoester
bond (overall spontaneous).
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20
 • Under alkaline (basic) conditions, RNA rapidly hydrolyzes.
• Ribose 2’ hydroxyl acts as nucleophile in intramolecular displacement.
• Might be why DNA is used for archiving genetic information instead of RNA.

residues 1 thru i

residue i

residues i+1 thru 3’ end

residue i+1

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 Nucleic acids form regular hydrogen bond patterns (secondary structure) via
base pairing. DNA exists mostly as fully base paired double helix (antiparallel
complementary strands). RNA, with its C2’ hydroxyl, is single-stranded and
forms more complicated secondary structures by folding back on itself.

• Watson-Crick “base pairing” occurs between purine:pyrimidine

in complementary polynucleotide strands.
– G:C
• 3 intermolecular hydrogen bonds. Higher melting temperature,
Tm, actually due to better pi stacking than:
– A:T (DNA)
• 2 hydrogen bonds (lower Tm)
– A:U (RNA)
• 2 hydrogen bonds

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 = -H in uracil
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Through-space (nonbonded) interactions in 3D structure
1. Hydrogen bonding between complementary bases (dashed);
2. Base stacking: parallel arrangement of bases.

Backbone: blue ribbon

Bases: yellow rectangles
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 Base Stacking
– Minimizes hydrophobic interactions
with water
– Bases are planar, aromatic
(conjugated p orbitals)
– Face-to-face pi (π-π) stacking
– Parallel-displaced (staggered) dipole-dipole interactions
• Mutagens: aromatic compounds (e.g. ethidium bromide) that
“intercalate” in between base pairs and disrupt DNA synthesis, causing
heritable genetic damage à intercalating agents. Mutations often
cause cancer (carcinogens).

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 Determinants of 3D structure in DNA and RNA

Backbone conformation
1. Slight sugar puckering (akin to
cyclohexane chair flipping). 3
2
2. Rotation of phosphodiester bonds.
3. Rotation of bonds connecting
pentose ring to phosphate groups. 3

1
Base
3
Stabilizing nonbonded interactions in
DNA and RNA. 2
• Hydrogen bonding (base pairs)
• Base (aromatic) stacking
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 Sugar puckering: note how the position of C3’ relative to C5’
changes; this changes the backbone conformation because the
3’, 4’, 5’ carbons are all in the backbone.

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 DNA is a Double Helix
• A duplex is formed between two strands of DNA with complementary
bases running in opposite directions:
5’-TGGAC-3’
3’-ACCTG-5’
• Double-stranded DNA forms a right-handed twist (like the threads on a
bolt).
– 3.4 Å between vertically stacked bases
– Helical repeat distance of 34-36 Å
(10 base pairs in each turn)
• Double helix contains a major groove:
– Base pair functional groups are exposed. Sequence-specific DNA-binding
proteins (transcription factors) recognize the major groove: zinc finger, helix-
turn-helix or leucine zipper domains.
• And a minor groove:
– Base pairs are more protected by the phosphate-sugar backbone. Binds
structural proteins (non-specific binding) that package DNA into compact
28 chromosomes.
 Watson and Crick model

Minor
groove

Major
groove

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 Replication of DNA
• The two strands of the parent
DNA double helix are unwound.
• Each strand acts as a template
to synthesize a new
complementary strand.
• Result is two identical copies of
DNA (“daughters” in cell
division).

Replication: Process of producing

two identical replicas of DNA from
one parent DNA.

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 Watson and Crick proposed
their model of DNA from 3 data:

1) Chargaff’s rules: [A] = [T]; [G] = [C];

[pyrimidine] = [purine].

2) Bases are relatively inaccessible and not titratable.

3) Rosalind’s X-ray diffraction image:

1962 Nobel Prize (Medicine)

to Watson, Crick, Wilkins.

“X” shape indicates helix.

Spacing indicates 3.4 and 34 Å
periodicity in long axis.

Rosalind was not on the Nobel because

the double helix was not considered
“proven science” until after her death.
 Double Helix Papers

Watson and Crick.

1953. A structure for
deoxyribose nucleic
acid. Nature 171:
737-8

Cambridge

King’s College, London

Wilkins, Stokes and Wilson. 1953.
Molecular structure of deoxypentose
nucleic acids. Nature 171: 738-40
32 Franklin and Gosling
 History of Structural Biology..

• Linus Pauling is considered the father of molecular

biology and is known for first popularizing vitamin C
with the general public.
• His discovery of sickle cell anemia as a “molecular
disease" paved the way for examining genetically
acquired mutations at a molecular level.
• 1949 at Caltech: sickle cell-disease is due to mutation
in specific protein (hemoglobin). 1954 Nobel Prize
in Chemistry
Pauling et al. 1949. Sickle cell anemia, a molecular
disease. Science 110: 543-8

• Pauling first proposed the alpha helix and beta sheet as the main
elements of secondary structure in proteins.

Pauling and Corey. 1951. Configurations of polypeptide chains with

favored orientations around single bonds: Two new pleated sheets.
Proc. Natl. Acad. Sci. USA 37: 729-40
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 Bases form a
spiral staircase
(not a ladder)

g ro o v e
m a j o r

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 DNA is a double helix
(secondary structure)

The Watson-Crick structure is called

B-form DNA
– Most stable structure for random-
sequence DNA under physiological
conditions
– Regular repeating structure can support:
• Long lengths; compact packaging (by coiling
DNA around spindle proteins: histones); good
information storage medium
• Other types of DNA double helices:
– “A-form” occurs when water is lacking
• Wider diameter than B-form (both are right-
handed)
– “Z-form” particular to certain sequences
• Left-handed; narrow diameter
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 Exceptions to the double helix
• Palindromic sequence repeats form intrastrand base pairs.
• Common in RNA.

Stem-loop structure 36
 Exception to duplex DNA: G-quadruplex

– Hydrogen bonding between 4 G’s in the same plane.

– Repetitive telomeric DNA (end of chromosome) is G-rich
and can loop back on itself, forming an intramolecular
quadruplex.
– The telomeres are normally degraded during each round of
replication (primer sequence can’t be replicated).

– Telomerase (implicated
in aging and cancer) can
extend telomeres (adds
TTAGGG repeats to the 3’
overhang), but the G-
quadruplexes must first be
unwound. Potential cancer
drugs stabilize the
quadruplex.
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 RNA: Transfer of information

• RNA takes on many structures.

• DNA à RNA: “transcription”
– Messenger RNA (mRNA) is synthesized on an unwound single strand DNA
template in the nucleus by RNA polymerase.
• RNA à protein: “translation”
– mRNA exits the nucleus and associates with ribosomes in the cytoplasm.
– mRNA + ribosome recruits transfer RNA (tRNA) covalently bound to an
amino acid at its 3’ hydroxyl.
– three adjacent bases on mRNA (codon) complement three adjacent bases
on tRNA (anticodon) à every three letters of the DNA sequence correspond
to an amino acid.
– mRNA streams through the ribosome; with each new tRNA that is recruited,
a new amino acid is added to the growing polypeptide chain through the
formation of a peptide bond.

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Three Steps of Protein Synthesis
 tRNA 3D Structure
Cloverleaf secondary structure:
local base-paired segments
(stem-loop hairpins)

Overall tertiary structure:

L-shaped stacked helices
fit into ribosome
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 Chromatin Structure

http://dx.doi.org/10.1126/science.caredit.a1000092

• Don and Ada (DnA) are current UNE

faculty. They discovered the basic
structural unit of chromatin, the
nucleosome, under the electron
microscope in 1974.

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 Nucleosomes are an example of
quaternary structure

• Histones are basic proteins that bind strongly to DNA. They help to
organize and compact DNA in the cell nucleus.
• They form nucleosomes, regular units of about 200 base pairs of DNA
wound around an octameric core of different histone proteins (beads
on a string).
• Nucleosomes can be packed together to form a coiled fiber.
• Conserved lysines can be acetylated (post-translational modification)
to loosen chromatin packing (amide is not charged and no longer
attracted to phosphate) and promote gene transcription.
• The pattern of chromatin packing can be transferred to offspring
(epigenetics). This is the basis of innate memory and tissue
differentiation during development.
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 Epigenetics

• Histone acetylation
− Histone acetyltransferases are enzymes that transfer an acetyl group
to conserved lysine residues on histone proteins, turning bound gene
sequences on or off. In general, histone acetylation increases gene
expression.

• DNA methylation
− Methylation can change the activity of a DNA segment without
changing the sequence. When located in a gene promoter, DNA
methylation typically acts to repress gene transcription. DNA
methylation is essential for normal development and is associated
with a number of key processes including genomic imprinting, X-
chromosome inactivation, repression of transposable elements, aging
and carcinogenesis.
 Nucleic acid chemistry:
Reversible heat denaturation and annealing

Double helix ! partially denatured DNA ! ssDNA

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 Strand Annealing
• Partially denatured DNA -> double helix (dsDNA) is fast because all that
needs to happen is for the DNA to finish “zipping” together.
• Single-stranded (ssDNA) -> partially denatured DNA is slow because the
separated ssDNA molecules must first find each other in solution and form
complementary base pairs (slow) before they can start zipping together
(fast).
• Monitored by light absorption:
– 260 nm light (ultraviolet “UV”) is absorbed by the aromatic bases.
– The bases are partially hidden by the backbone (and more ordered) in dsDNA.
ssDNA has its bases exposed to solvent.
– dsDNA absorbs less 260 nm UV than ssDNA. Free nucleotides absorb even more
than ssDNA.

• dsDNA rich in G:C pairs denature at higher temperatures than A:T regions.
• Sites of DNA replication and transcription tend to be A:T rich. Upstream gene
promoter sequence: 5’-TATAAA-3’ (TATA box).
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 Partially Denatured DNA

single-stranded
bubbles

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 Nucleic acid chemistry:
Hybridization

• Used to detect presence of a particular DNA

sequence in a mixture (e.g. forensics).
– Need a labeled sample (e.g. with radioisotope, 33P) with
sequence complementary to the one you wish to detect.
– Heat the labeled sample and the unknown mixture to fully
denature DNA in both samples and create ssDNA.
– Mix the two samples and gradually cool.
– If the sequence of interest exists in the unknown mixture,
it will hybridize (form a double helix with) a strand of the
labeled complementary sample.
– Hybrid duplex can then be detected.
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 Nucleic acid chemistry: Non-enzymatic covalent
transformations (DNA damage)

• Spontaneous damage
– Oxidative deamination – loss of amino groups
• C -> U
• 5-methyl-C -> T
• A -> hypoxanthine
• G -> xanthine (xanthine derivatives are stimulants)
– Depurination: N-glycosidic bond between A or G base and
deoxyribose is hydrolyzed à base floats away. Most
frequent type of DNA lesion; leads to cancer mutations.

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49
 • UV damage
UV light-induced
3.4 Å
dimerization of adjacent
pyrimidine bases causes
skin cancer.

1.5 Å

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 • Chemical damage (e.g. from chemicals in the environment, in food,
or in the organism itself).
– Deamination (accelerated relative to spontaneous deamination)
• Nitrous acid and other food preservatives.
– Alkylation
– Oxidation
• Oxidative chemicals form both from irradiation and from
aerobic metabolism.
• Cellular enzymes exist to convert these to harmless
compounds.
• DNA repair enzymes can correct damage that occurs in only one of the
strands in the double helix, but are not 100% efficient.
• Unrepaired DNA damage can lead to aging in non-replicating cells, or in
replicating cells, cancer.
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 Mutations occur during replication as a result of a mismatch in base pairing,
caused by tautomerization, protonation, or chemical agents (oxidation).

X
s
is r upt
d s
- b ond
H

(Watson-Crick pair)

When caused during the normal course of metabolism, this leads to

a background mutation rate of naturally occurring DNA damage.
52
 Mutagens/carcinogens in Food
• Polycyclic aromatics are intercalating agents that cause genetic mutation.
They are created in curing processed meat or by high temperature pan-
frying or grilling of red meat.
http://www.thelancet.com/pdfs/journals/lanonc/PIIS1470-2045(15)00444-1.pdf

• Dioxins accumulate up the food chain and can be formed by microwaving

food in contact with plastics.
http://www.ejnet.org/dioxin/dioxininfood.pdf

dioxin

http://www.whatthehealthfilm.com/facts
 Other carcinogens in food

• The browning of bread, meat and roasted coffee get their odor
and flavor from a mixture of compounds that result from amino
acids reacting with sugars at elevated temperatures (the Maillard
reaction).
• The sequence of events starts with the nucleophilic amino group
(enhanced in basic solution) attacking the sugar carbonyl group
to form a glycosylamine (β-N-glycoside). At high temperatures,
products can include cancer-causing acrylamide and furans.
• The Maillard reaction also takes place spontaneously in human
tissue, and its products are linked to diabetes and cataracts.

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 Fluorouracil - Pharmacology

• 5-fluorouracil (5-FU) is a WHO essential medicine used by

injection to treat colon and other types of cancer; as a cream
it is used to treat basal cell carcinoma (skin cancer).
• Pyrimidine antimetabolites are a class of cancer medications
(analogs of the metabolic pyrimidines) whose mechanism is
interfering with DNA replication.
• 5-FU block DNA production in dividing cells by inhibiting
thymidylate synthase, which normally is responsible for
making deoxythimidine monophosphate (dTMP) from dUMP
(uracil nucleotide).
• 5-FU is first converted by the body into the active metabolite 5-fluoro
2’-deoxyuridine monophosphate (F-dUMP). 5-dUMP inhibits the
synthase enzyme’s active site.
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 Concept map
for DNA
structure

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