Nucleotides and Nucleic Acids
Chapter 8
Nucleotides & Nucleic Acids
Nucleotides are the building blocks of nucleic acids
A nitrogenous base
A phosphate group
(pyrimidines or purine)
A pentose sugar
Nucleotides have three characteristic components
Chapter 8
Structure of a Nucleotide
We Need Nucleic Acids!
The Ribose Sugar
DNA
Protein Protein
Trait
DNA RNA
mRNA
RNA
rRNA
Pol
tRNA
3
• DNA contains genes, the information needed to synthesize functional proteins
and RNAs
– DNA also contains segments that play a role in regulation of gene
expression (promoters, operators, etc.)
• Messenger RNAs (mRNAs) are transcribed from DNA by RNA polymerases
and carry genetic information from a gene to the ribosome complex
• Ribose (β-Dribofuranose) is a
pentose sugar
• Note numbering of the
carbons.
• In a nucleoside or
nucleotide, "prime" is
used (to differentiate
from base numbering).
• Ribosomal RNAs (rRNAs) are components of the ribosomes and play a role
in protein synthesis in conjunction with the template mRNA and the AA
carrying tRNA
5
4
1
3
2
• Transfer RNAs (tRNAs) carry the AAs designated by the codons of the
mRNA and bind to the Ribosome to help form the growing polypepide chain
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Structure of a Nucleotide
Structure of a Nucleotide
The Pyrimidine and Purine Bases
Bases from the minor leagues…
Big word, small ring
Small word, big ring
Know these:
Numbering System and General Ring Structure
Chapter 8
5
• 5-Methylcytidine occurs in DNA of animals and higher plants
• N6-methyladenosine occurs in bacterial DNA
Chapter 8
These 8 bases you do not need to know…
Structure of a Nucleotide
Structure of a Nucleotide
Major Bases in Nucleic Acids
The Phosphate
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• The bases are abbreviated by
their first letters (A, G, C, T, U).
• The purines (A, G) occur in
both RNA and DNA
• The pyrimidine C occurs in
both RNA and DNA, but
• T occurs only in DNA, and U
occurs only in RNA
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Be able to recognize and draw these five!
A nucleoside + one or more phosphoryl
groups is called a nucleotide.
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Structure of a Nucleotide
Structure of a Nucleotide
Linkages
Phosphate Variations: Number
The phosphate
at the 5’ carbon
Sugar to the
base at 1’ carbon
1
2
3
Note the hydroxyl
at the 2’ carbon
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Why are these important?
Nucleoside mono-, di-, or triphosphate
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Chapter 8
Structure of a Nucleotide
Structure of a Nucleotide
Nucleotides in Nucleic Acids
Ribonucleotides
• The ribose sugar with a base
(here, a pyrimidine, uracil or
cytosine) attached to the
ribose C-1' position is a
ribonucleoside (here, uridine
or cytidine).
• Bases attach in β-linkage to
the C-1' of ribose or
deoxyribose
• The pyrimidines attach to the
pentose via the N-1 position
of the pyrimidine ring
• Phosphorylate the 5' position
and you have a
ribonucleotide (here,
uridylate or cytidylate)
• The purines attach through
the N-9 position
• Some minor bases may have
different attachments.
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• Ribonucleotides are abbreviated (for example) U, or UMP
(uridine monophosphate)
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Structure of a Nucleotide
Structure of a Nucleotide
Deoxyribonucleotides – Better for DNA!
The Major Ribonucleotides
• A 2'-deoxyribose sugar with a
base (here, a purine - adenine
or guanine) attached to the C1' position is a
deoxyribonucleoside
• Phosphorylate the 5' position
and you have a nucleotide
• Deoxyribonucleotides are
abbreviated (for example) A,
or dA or dAMP
Note the N9 (purine) and N1 (pyrimidine)
linkage to the ribose ring at C1’
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Structure of a Nucleotide
Structure of a Nucleotide
One of RNA’s Little Problems – Stability!
The Major Deoxyribonucleotides
DNA, which lacks a
2’-OH, is stable
under alkaline
conditions…
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Structure of a Nucleotide
Structure of a Nucleotide
Now, on to Nucleic Acids!
Nucleotide Nomenclature
• Nucleotide monomers can be
linked together via a
phosphodiester linkage
formed between the 3' -OH of
a nucleotide and the
phosphate of the next
nucleotide.
• Two ends of the resulting
poly- or oligonucleotide are
defined:
– The 5' end lacks a nucleotide
at the 5' position
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Chapter 8
– The 3' end lacks a nucleotide
at the 3' end position
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Structure of a Nucleotide
Structure of a Nucleotide
Nucleotide Nomenclature
Sugar-Phosphate Backbone
Berg et al. “Biochemistry”
5th Ed. Fig. 1.1
• The polynucleotide or nucleic acid backbone thus consists of
alternating phosphate and pentose residues.
• The bases are analogous to side chains of amino acids; they vary
without changing the covalent backbone structure.
• Sequence is written from the 5' to 3' end: 5'-ATGCTAGC-3'
• Note that the backbone is polyanionic, and the phosphate groups have
a pKa ~ 0.
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Structure of a Nucleotide
Nucleic Acid Structure
The bases can take syn- or anti- positions
Polynucleotides vs. Polypeptides
• As in proteins, the sequence of side chains
(bases in nucleic acids) plays an important
role in function.
• Nucleic acid structure depends on the
sequence of bases and on the type of
ribose sugar (ribose, or 2'-deoxyribose).
• Hydrogen bonding interactions are
especially important in nucleic acids, both
interstrand and intrastrand.
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Structure of a Nucleotide
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Nucleic Acid Structure
Sugar phosphate backbone conformation
DNA
• Polynucleotides have unrestricted
rotation about most backbone bones
(within limits of sterics)
• One exception is the sugar ring bond
Chapter 8
• DNA consists of two helical
chains wound around the same
axis in a right-handed fashion
aligned in an antiparallel
fashion.
• This behavior contrasts with the
peptide backbone.
• There are 10.5 base pairs, or 36
Å, per turn of the helix.
• Also in contrast with proteins, specific,
predictable interactions between bases
are often formed: A with T, and G with
C.
• Alternating deoxyribose and
phosphate groups on the
backbone form the outside of the
helix.
• These interactions can be interstrand,
or intrastrand.
• The planar purine and
pyrimidine bases of both strands
are stacked inside the helix.
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Nucleic Acid Structure
Nucleic Acid Structure
DNA
Stabilization of Double Helix
• Weak forces stabilize the double helix:
• The furanose ring usually is
puckered in a C-2' endo
conformation in DNA.
(1) Hydrophobic Effects: Burying purine and pyrimidine
rings in the interior of the helix excludes them from water
• The offset of the relationship of
the base pairs to the strands
gives a major and a minor
groove.
(2) Stacking interactions: Stacked base pairs form van der
Waals contacts
(3) Hydrogen Bonds: H-bonding between the base pairs (not
on the backbone!)
• In B-form DNA (the most
common) the depths of the major
and minor grooves are similar to
each other.
(4) Charge-charge interactions: Electrostated repulsion of
negatively charged phosphate groups is decreased by
cations (e.g. Mg2+) and cationic proteins
• The base-pairs are perpendicular
to the helical axis
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Nucleic Acid Structure
Nucleic Acid Structure
DNA Strands
Interstrand H-bonding Between DNA Bases
• The opposing strands of DNA are not identical, but
are complementary.
• This means: they are positioned to align
complementary base pairs:
– C with G, and A with T, but the strands run antiparallel to
each other
• You can thus predict the sequence of one strand
given the sequence of its complementary strand.
• Note that sequence conventionally is written from
the 5' to 3' end
• Such a structure is useful for information storage
and transfer!
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Watson-Crick base pairing
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Nucleic Acid Structure
Nucleic Acid Structure
Base Stacking in DNA
DNA Structure Summary
• C-G (red) and A-T (blue) base
pairs are isosteric (same shape
and size), allowing stacking along
a helical axis for any sequence.
Chapter 8
• Base pairs stack inside
the helix.
Berg et al. “Biochemistry” 5th Ed. Figs. 1.4; 5.13
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Nucleic Acid Structure
Nucleic Acid Structure
Various Nucleic Acid Structures
Unusual DNA Structures
• B form - The most common
conformation for DNA.
• A form - common for RNA
because of different sugar
pucker. Deeper minor groove,
shallow major groove
36 base pairs
Backbone - blue;
Bases- gray
– A form is favored in conditions of low
water.
• Some structures seen in DNA are
sequence dependent
• Z form - narrow, deep minor
groove. Major groove hardly
existent. Can form for some DNA
sequences; requires alternating
syn and anti base configurations.
Chapter 8
• These structures can be very
important in the binding of
proteins to the nucleic acid
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Nucleic Acid Structure
Nucleic Acid Structure
RNA
Large RNAs may be highly structured
• In gene expression, RNA acts as an intermediary between the
genetic information of DNA and the production of polypeptides
• The 337 nt long M1 RNA of the tRNA
processing enzyme RNase P of E. coli has
many hairpin loops
• Even for a random RNA sequence, about
60-70% may be involved in secondary
structure (why?)
• In eukaryotes, DNA is largely confined to the nucleus whereas
protein production occurs in the cytoplasm.
• RNA allows these two processes to remain separated by
shutting between them
• The product of transcription is always a single stranded RNA
G-U pairs
– Tends to assume a right handed helical conformation
dominated by base-stacking interactions
G-U pairs are also
allowed, as they
are energetically
neutral.
• Any self-complimentary sequences or interactions with a
second RNA or a DNA chain will lead to duplex formation with
one difference from duplex DNA: Uracil instead of Thymine
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Nucleic Acid Structure
Nucleic Acid Structure
RNA has a Rich and Varied Structure
RNA Displays Interesting Tertiary Structure
• Watson-Crick base pairs
in helical segments
(usually A-form).
Singlestranded RNA
right-handed
helix
• Helix is secondary
structure.
Note A-U pairs in RNA
Green =
phosphodiester
backbone
DNA can also
form structures
like this.
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Other regions may also pair,
which facilitates tertiary folding.
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Yeast tRNAPhe
(1TRA)
Hammerhead ribozyme
(1MME)
T. thermophila intron,
A ribozyme (RNA
enzyme) (1GRZ)
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
• Replication: The preparation of exact copies of a molecule.
DNA replication is essential for life, and is what allows
organisms to create copies of themselves.
• Transcription: The copying of the genetic message
encoded in DNA to RNA
• Translation: The translation of the genetic message
encoded in RNA into a polypeptide with a specific amino
acid sequence
• In DNA replication, each DNA strand
serves as a template for the
synthesis of a new strand, producing
two DNA molecules, each with one
new strand and one old strand.
• This is termed semiconservative
replication.
• Performing these functions requires transformations of these
complex molecules to occur without losing or damaging the
genetic information (i.e. causing mutations).
• Thus, chemical modifications of nucleic acids must be
prevented, controlled, monitored, and fixed if needed.
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DNA Replication
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• Replication requires separation of the
DNA strands (transient melting, or
denaturation) so that the parent
strands can serve as templates.
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Transformations of Nucleic Acids
Partially Denatured DNA
• DNA strands can be separated (DNA
denaturation) by increasing temperature.
• For DNA and RNA to perform their respective and various
functions within organisms, they must undergo physical and
chemical changes themselves
Physical
• Denaturation is a result of disruption of
interactions that stabilize DNA structure:
Hydrogen-bonding
Denaturation (Melting)
Renaturation (Annealing)
Shearing
Base stacking
• This electron micrograph shows DNA that has
been partially denatured with heat, and then
fixed.
Chemical
Chapter 8
Replication, synthesis
Cleavage
Deamination
Depurination
Crosslinking
Alkylation
Oxidation
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• The red arrows point to the single-stranded
regions, which appear as “bubbles” in the doublestranded backbone.
• Would you expect these regions to be the same
or different each time you denature this piece of
DNA? Why or why not?
• What characteristics would these regions have?
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Annealing: Hybridization
DNA Denaturation and TM
• Experiment: completely denature
duplex DNAs from a fly and a human.
• Since DNA denaturation is commonly
done by increasing temperature, it is
also called DNA melting.
• Mix the denatured samples and keep at
~ 65 ˚C for many hours
• This can be followed by observing the
change in the ultraviolet absorption
band of DNA.
• Under these conditions, complementary strands will anneal.
• Most fly strands anneal with fly; and
human with human
• Stacked DNA bases absorb less light
than unstacked; this is called
hypochromism.
• But some strands of fly and human
DNA will anneal to give hybrid
duplexes.
• The "melting temperature" Tm is taken
as the inflection point of the melting
curve.
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• This reflects a common evolutionary
heritage.
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
DNA Melting and Annealing
Nucleic Acids Can Be Chemically Altered
• DNA denaturation is reversible.
• The formation of double helical DNA
from denatured DNA is called
annealing.
Deamination
Depurination
• Lowering the temperature below Tm
(given the right pH, ionic strength, etc.)
will allow annealing.
Crosslinking
Alkylation
Tm depends on pH, ionic
strength, DNA length, and
DNA base composition
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What would be different if this
were done with human and
chimp DNA?
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Oxidation
What will result from this…?
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Base Deamination
Mutation by Deamination
• Nucleotide bases can undergo
spontaneous loss of their exocyclic amino
groups (deamination).
• Under typical cellular conditions,
deamination of cytosine in DNA to uracil
occurs in about one of every 107 residues
every 24 hours.
Uracil
• A and G deamination occurs at 1/10 of this
rate.
Can now
pair with
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Mutation by Deamination
Thymine in DNA Allows Error Correction!
• Why is U present in RNA, and T in DNA?
• Deamination of Cytosine is mutagenic.
• When deaminated, cytosine becomes uracil which pairs
with A (rather than G)
• This “mispairing” causes a G to A substitution in the
complementary strand.
• Spontaneous deamination of U, which gives C, is relatively common.
• This is potentially mutagenic
• The enzyme uracil glycosylase recognizes U in DNA and cleaves the
glycosidic bond to remove the base.
• Mutant cells lacking uracil glycosylase have a high rate of G-C to A-T
base pair mutations.
Uracil
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
UV Damage to DNA
Depurination
• Formation of a cyclobutane pyrimidine
dimer introduces a bend or kink into the
DNA helix
• The N-β-glycosyl bond between
the base and the pentose can
undergo hydrolysis.
• 50-100 of these lesions occur in each skin
cell for every second of sun exposure(!)
• This process is faster for purines
than for pyrimidines
• Most of these are recognized and
repaired immediately.
• 1/100,000 purines are lost from
DNA every 24 hours
• If damage goes uncorrected, permanent
mutation may result.
• Depurination of RNA and of
ribonucleotides is much slower.
Chapter 8
• CC to TT mutation occurs if a CC dimer is
mispaired with two adenine bases. This is
often seen in the p53 gene in skin
cancers.
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Crosslinking of Bases
Mutation by Methylation
• Pyrimidine dimers can be
formed by exposure to UV light.
• Methylation of the guanine
carboxyl oxygen results in
the formation of a
nucleotide that can only
form two hydrogen bonds
with its partner
• Most commonly seen for
adjacent thymidine residues on
the same DNA strand
• Cyclobutane, or 6-4-linked
dimers, can be formed.
• This methylated gaunine
can then be pair with T
rather than C
• This causes a kink in the helix
• X-rays and gamma rays can
cause ring opening and base
fragmentation.
Chapter 8
Chapter 8
• This is a weaker pair in
addition to a mutagenic
pairing
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Nucleic Acid Chemistry
Nucleic Acid Chemistry
Automated DNA Sequencing
Chemical Alkylating Agents – Harmful to Children
and Other Living Things…
• One major improvement in recent years has been the development of automated procedures for
fluorescent DNA sequencing (Wilson et al., Genomics, 1990, pg. 626).
• These procedures generally use primers or dideoxynucleotides to which are attached fluorophores
(chemical groups capable of fluorescing).
• During electrophoresis, a monitor detects and records the fluorescence signal as the DNA passes through
a fixed point in the gel.
• The use of different fluorophores in the four base-specific reactions means that, unlike conventional DNA
sequencing, all four reactions can be loaded into a single lane.
• The output is in the form of intensity profiles for each of the differently colored fluorophores, but the
information is simultaneously stored electronically.
• Nitrogen mustard. First symptoms of exposure: skin, eye and
respiratory tract irritation, burns and blistering.
• Long-term effects include leukemia.
• Used as a treatment of Hodgkin's disease and brain tumors (!)
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Figure 6.17 from Human Molecular Genetics, 2nd Ed., Strachan et al.
Nucleic Acid Chemistry
Nucleic Acid Chemistry
Chemical Synthesis of DNA
DNA Sequencing: Dideoxy Method
(A) The Dideoxy method relies on the insertion of
ddNTPs into a growing DNA chain, which causes
chain termination.
(A) NTP is attached to the silica
support
(B) DMT protecting group is
removed
(B) Purified target DNA is synthesized using a primer
specific for the strand to be sequenced.
(C) The incoming NTP is activated
and coupled to the immobilized
NTP to form a 5’ to 3’ linkage
(C) In four separate reactions, four different ddNTP’s are
added into the reaction mixture (in addition to the
normal dNTP’s) resulting in the formation of DNA
strands of varying lengths.
(D) The dinucleic acid is oxidized to
produce the phosphotriester
(D) These strands can be separated using
electrophoresis and the sequence read by comparing
the lanes.
(E) Steps (B) through (D) are
repeated
(F) The remaining protecting
groups are removed
Sequencing
Animation
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(G)The oligonucleotide is
separated from the support
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Chapter 8
Why would synthesis of RNA be more difficult?
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Other Functions of Nucleotides
• Building blocks of nucleic
acids (RNA, DNA)
(analogous to amino acid
role in proteins)
• Energy currency in cellular
metabolism (ATP:
adenosine triphosphate)
• Allosteric effectors
• Structural components of
many enzyme cofactors
(NAD: nicotinamide adenine
dinucleotide)
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