Reginald H. Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett
Chapter 11
Structure of Nucleic Acids
Reginald Garrett & Charles Grisham • University of Virginia
Outline
• How do scientists determine the primary structure of
nucleic acids?
• What sorts of secondary structures can double-stranded
DNA molecules adopt?
• Can the secondary structure of DNA be denatured and
renatured?
• Can DNA adopt structures of higher complexity?
• What is the structure of eukaryotic chromosomes?
• Can nucleic acids be synthesized chemically?
• What are the secondary and tertiary structures of RNA?
11.1 How Do Scientists Determine the
Primary Structure of Nucleic Acids?
• Two simple tools have made nucleic acid
sequencing easier than polypeptide sequencing:
• The type II restriction endonucleases that cleave
DNA at specific oligonucleotide sites
• Gel electrophoresis, which is capable of
separating nucleic acid fragments that differ from
one another in length by just a single nucleotide
11.1 How Do Scientists Determine the
Primary Structure of Nucleic Acids?
• Chain termination method (dideoxy method),
developed by Frederick Sanger is the basis for
most DNA sequencing currently.
• The method takes advantage of the DNA
polymerase reaction, which copies a DNA strand
in complementary fashion to form a new second
strand
11.1 How Do Scientists Determine the
Primary Structure of Nucleic Acids?
• DNA is a double-helical molecule
• Each strand of the helix must be copied in
complementary fashion by DNA polymerase
• Each strand is a template for copying
• DNA polymerase requires template and
primer
• Primer: an oligonucleotide that pairs with the
end of the template molecule to form dsDNA
• DNA polymerases add nucleotides in 5'-3'
direction
11.1 How Do Scientists Determine the
Primary Structure of Nucleic Acids?
DNA replication yields two daughter DNA
duplexes identical to the parental DNA
molecule.
Chain Termination Method
• Primer extension: A template DNA base-paired
with a complementary primer is copied by DNA
polymerase in the presence of dATP, dCTP,
dGTP, dTTP
• Solution contains small amounts of the four
dideoxynucleotide analogs of these substrates,
each of which contains a distinctive fluorescent
tag, illustrated here as:
• Orange for ddATP
• Blue for ddCTP
• Green for ddGTP
• Red for ddTTP
Occasional incorporation of a dideoxynucleotide
terminates further synthesis of that strand
The chain termination
method of DNA
sequencing.
Chain Termination Method
• Most of the time, the polymerase uses normal
nucleotides and DNA molecules grow
normally
• Occasionally, the polymerase uses a
dideoxynucleotide, which prevents further
extension when added to the growing chain
• Random insertion of dd-nucleotides leaves
(optimally) at least a few chains terminated at
every occurrence of a given nucleotide
Chain Termination Method
• Reaction mixtures can be separated by capillary
electrophoresis
• Short fragments go to bottom, long fragments on
top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary
sequence
• Now read from the other end and you have the
sequence you wanted - read 5' to 3'
The set of terminated strands can be
separated by capillary electrophoresis
Emerging Technologies to Sequence DNA are
Based on Single-Molecule Sequencing Strategies
• Growing demand for sequence information is driving
the development of faster and cheaper methods of
DNA sequencing
• Most promising are the single-molecule strategies that
do not rely on Sanger-based primed synthesis of
strands complementary to prepared DNA samples
• One technique involves passing a single strand of
DNA through a graphene monolayer pore, measuring
the change in electrical conductance (ion flow)
through the pore
• Each base alters electrical conductance in a subtle
but different way, facilitating the “reading” of
sequence
Figure 11.5 DNA Sequencing through a pore in a
graphene monolayer
11.2 What Sorts of Secondary Structures Can
Double-Stranded DNA Molecules Adopt?
The six degrees of freedom in the deoxyribose-PO4 units
of the polynucleotide chain. The seventh free rotation is
about the C1'-N glycosidic bond.
DNA structure
(a) Double-stranded DNA as an imaginary
ladderlike structure.
(b) A simple right-handed twist converts the
ladder to a helix.
11.2 What Sorts of Secondary Structures Can
Double-Stranded DNA Molecules Adopt?
• The stability of the DNA double helix is due to:
• Hydrogen bonds – between base pairs
• Electrostatic interactions – mutual repulsion of
phosphate groups, which makes them most stable
on the helix exterior
• Base-pair stacking interactions
• Right-twist closes the gaps between base pairs to
3.4 A (0.34 nm) in B-DNA
The “canonical” base pairs
• The canonical A:T and G:C base pairs have
nearly identical overall dimensions
• A and T share two H bonds
• G and C share three H bonds
• G:C-rich regions of DNA are more stable
• Polar atoms in the sugar-phosphate backbone
also form H bonds
Major and minor grooves
Major and minor grooves
• The "tops" of the bases (as we draw them) line
the "floor" of the major groove
• The major groove is large enough to
accommodate an alpha helix from a protein
• Regulatory proteins (transcription factors) can
recognize the pattern of bases and the H-bonding
possibilities in the major groove
The “canonical” base pairs
Watson-Crick A:T and
G:C base pairs. All Hbonds in both base
pairs are straight.
Double Helical Structures Can Adopt a
Number of Stable Conformations
• The DNA double helix can adopt several stable
conformations
• Helical twist is the rotation of one base pair relative
to the next, around the axis of the double helix
• Successive base pairs in B-DNA show a mean
rotation of 36º with respect to each other
• Propellor twist involves rotation around a different
axis, namely an axis perpendicular to the helix axis
Double Helical Structures Can Adopt a
Number of Stable Conformations
(a) Helical twist: Successive base pairs in B-DNA show a
rotation with respect to each other.
Double Helical Structures Can Adopt a
Number of Stable Conformations
Figure 11.10 Helical twist and propellor twist in DNA.
(b) Propellor twist: Rotation in this dimension allows the
hydrophobic surfaces of bases to overlap better
Double Helical Structures Can Adopt a
Number of Stable Conformations
Each of the bases in a base pair shows positive propellor
twist as viewed along the N-glycosidic bond. Note how the
hydrogen bonds between bases are distorted by this motion,
yet remain intact.
Double Helical Structures Can Adopt a
Number of Stable Conformations
The B-form of the DNA double
helix. In B-form, the pitch (the
distance required to complete one
helical turn) is 3.4 nm. Twelve
base pairs of DNA are shown.
Double Helical Structures Can Adopt a
Number of Stable Conformations
The A-form of the DNA
double helix. The pitch of the
A-form helix is 2.46; thus the
A-form is a shorter, wider
structure than the B-form.
One turn in A-form DNA
requires 11 bp to complete.
Twelve base pairs are shown
here.
Z-DNA
•
•
•
•
Discovered by Alex Rich
Found in G:C-rich regions of DNA
G goes to syn conformation
C stays anti but whole C nucleoside (base and
sugar) flips 180 degrees
Result is that G:C H bonds can be preserved
in the transition from B-form to Z-form!
Z-DNA is a Conformational Variation in the
Form of a Left-Handed Double Helix
• The Z-form of double helical DNA.
• The N-glycosyl bonds of G
residues in this alternating
copolymer are rotated 180º with
respect to their conformation in BDNA, so now the G nucleoside is in
the syn rather than the anti
conformation.
• The C residues remain in the anti
form.
• Because the G ring is flipped, the C
ring must also flip to maintain
normal Watson-Crick base pairing.
Comparison of A, B, Z DNA
• A: right-handed, short and broad, 2.3 Å, 11 bp
per turn
• B: right-handed, longer, thinner, 3.32 Å, 10 bp
per turn
• Z: left-handed, longest, thinnest, 3.8 Å, 12 bp
per turn
DNA Methylation and Epigenetics
• Methylation of cytosine residues (forming 5methylcytosine) is essential for normal embryonic
development
• Cytosine methylation switches genes off, so that the
information they encode is not expressed
• Epigenetics is the study of heritable changes in the
genome that occur without a change in nucleotide
sequence (such as cytosine methylation)
• Epigenetic changes can influence expression of the
information encoded by the genome
Intercalating Agents Distort the Double
Helix
• The double helix is a very dynamic structure
• Because it is flexible, aromatic macrocycles – flat
hydrophobic molecules composed of fused,
heterocyclic rings, can slip between the stacked
pairs of bases
• The bases are force apart to accommodate these
intercalating agents
• Ethidium bromide
• Acridine orange
• Actinomycin D
Alternative H-Bonding Interactions Give
Rise to Novel DNA Structures
Cruciform structures arise from
inverted repeats. In such structures,
the normal interstrand base pairing is
replaced by intrastrand pairing.
Self-complementary inverted repeats can
rearrange to form H-bonded cruciform stemloop structures. Cruciforms are not as stable
as normal DNA, because an unpaired
segment must exist in the loop.
11.3 Can the Secondary Structure of DNA
Be Denatured and Renatured?
• When DNA is heated to 80°C or more, its UV
absorbance increases by 30-40%
• This hyperchromic shift reflects the unwinding
of the DNA double helix
• Stacked base pairs in native DNA absorb less
light due to p,p electron interactions
• When T is lowered, the absorbance drops,
reflecting re-establishment of the double helix
and base-pair stacking
The Buoyant Density of DNA
Density gradient ultracentrifugation is
a useful way to separate and purify
nucleic acids.
The net movement of solute particles in an ultracentrifuge is
the result of two processes: diffusion (from regions of higher
concentration to regions of lower concentration) and
sedimentation due to centrifugal force.
Single-Stranded DNA Can Renature to
Form DNA Duplexes
• Denatured DNA will renature to re-form the duplex
structure if the denaturing conditions are removed
• Renaturation requires reassociation of the DNA
strands into a double helix, a process termed
reannealing
• For this to occur, the strands must realign so that
their complementary bases are once again in
register and the helix can be “zippered up”
Single-Stranded DNA Can Renature to
Form DNA Duplexes
Nucleic Acid Hybridization: Different DNA Strands
of Similar Sequence Can Form Hybrid Duplexes
• If DNA from two different species are mixed,
denatured, and allowed to cool slowly, hybrid
duplexes may form, provided the DNA from one
species is similar in sequence to the other
• The degree of hybridization is a measure of the
sequence similarity between the two species
• 25% of the DNA from a human forms hybrids with
mouse DNA, implying some sequence similarity
• Hybridization is a common procedure in molecular
biology for identifying specific genes and for
revealing evolutionary relationships
11.4 Can DNA Adopt Structures of Higher
Complexity?
• In duplex DNA, there are ten bp per turn of helix
• Circular DNA sometimes has more or less than 10
bp per turn - a supercoiled state
• Enzymes called topoisomerases or gyrases can
introduce or remove supercoils
• Cruciforms occur in palindromic regions of DNA
• Negative supercoiling may promote cruciforms
Supercoils Are One Kind of Structural
Complexity in DNA
Double-stranded circular DNA forms supercoils, if the
strands are underwound, or overwound.
11.5 What Is the Structure of Eukaryotic
Chromosomes?
• Human DNA’s total length is ~2 meters!
• This must be packaged into a nucleus that is
about 5 micrometers in diameter
• This represents a compression of more than
100,000!
• It is made possible by wrapping the DNA
around protein spools called nucleosomes and
then packing these in helical filaments
• These filaments are thought to arrange in loops
associated with the nuclear matrix
Structural Organization of Chromatin Gives Rise to
Chromosomes
• The beads-on-a-string motif is the “primary” structure of
chromatin.
• The “secondary” level of chromatin structure is the 30-nm
fiber, formed when an array of nucleosomes in a zig-zag
pattern adopts a two-start helical conformation (Figures
11.29a, b, c).
• Higher levels of chromatin structural organization are
achieved when the 30-nm fiber forms long loops of 60150,000 bp.
• Electron microscopic analysis of human chromosome 4
suggests that 18 such loops are then arranged radially
about the circumference of a single turn to form a
miniband unit of the chromosome.
11.7 What Are the Secondary and Tertiary
Structures of RNA?
• The double-stranded structure of DNA imposes great
constraints on its conformational possibilities
• RNA molecules are typically single-stranded and
thus have six to seven degrees of freedom per
nucleotide unit
• Thus RNA molecules have a much greater number
of conformational possibilities
• Complementary sequences in RNA can join via
intrastrand base pairing
• When the base pairing is not complete, a variety of
bulges and loops can form, including hairpin stemloop structures
11.7 What Are the Secondary and Tertiary
Structures of RNA?
Bulges and
loops formed in
RNA when
aligned
sequences are
not fully
complementary
11.7 What Are the Secondary and Tertiary
Structures of RNA?
• A number of defined structural motifs recur within the
loops of stem-loop structures, such as U-turns,
tetraloops, and bulges
• Regions where several stem-loop structures meet
are termed junctions
• Stems, loops, bulges, and junctions are the four
basic secondary structural elements in RNA
• Other tertiary structural motifs arise from coaxial
stacking, pseudoknot formation, and ribose
zippers
11.7 What Are the Secondary and Tertiary
Structures of RNA?
Junctions and coaxial stacking in RNA.
11.7 What Are the Secondary and Tertiary
Structures of RNA?
RNA pseudoknots are
formed when a singlestranded region of RNA
base-pairs with a hairpin
loop.
Transfer RNA Adopts Higher-Order
Structure Through Intrastrand Base Pairing
• In tRNA, with 73-94 nucleotides in a single
chain, a majority of the bases are hydrogenbonded to one another
• Hairpin turns bring complementary stretches of
bases into contact
• Extensive H-bonding creates four double helical
domains, three capped by loops, one by a stem
• Only one tRNA structure (alone) is known
• Phenylalanine tRNA is "L-shaped"
• Many non-canonical base pairs found in tRNA
tRNA Tertiary Structure Arises From
Interloop Base Pairing
The three-dimensional
structure of yeast
phenylalanine tRNA.
The anticodon loop is
at the bottom and the
acceptor end is at the
top right.
Ribosomal RNA
•
•
•
•
•
Ribosomes synthesize proteins
All ribosomes contain large and small subunits
rRNA molecules make up about 2/3 of ribosome
High intrastrand sequence complementarity leads
to extensive base-pairing
Secondary structure features seem to be
conserved, whereas sequence is not
There must be common designs and functions
that must be conserved
Ribosomal RNA also Adopts Higher-Order
Structure Through Intrastrand Base Pairing
These secondary structures of several 16S rRNAs are based on
computer alignment of rRNA nucleotide sequences into optimal
H-bonding segments.
Comparison of secondary structures of 16S-like rRNAs from
several organisms.
Riboswitches Act as Regulators of Gene
Expression
Riboswitches, naturally
occurring aptamers, are
conserved regions of mRNAs
that reversibly bind specific
metabolites and coenzymes
and act as gene expression
regulators.
End of Chapter Questions
• While all of the questions will make you think about
the material, 1-5 are the only ones to focus on.