Lecture 6 – Nucleic Acid

Structure
Introduction
The term, ‘Nucleic Acid’:
 refers to the functional forms of polynucleotides.
Nucleic acid structure:
 follows many of the principles we learned for
polypeptides.
Some important differences, however:
1. fewer building blocks:
 each type constructed from only 4 types of
monomers…
 for a given length, fewer molecules can be
constructed.
 each monomer has many more torsion angles:
 polynucleotide chains much more flexible.
 These differences will affect the number of different
Nucleotides
The monomer building blocks of Nucleic Acids
are Nucleotides.
 All have a D-stereoisomeric configuration.
 Each nucleotide consists of:
 a phosphate (PO4-),
 attached to the 5’ Carbon = 5’ nucleotide.
 attached to the 3’ Carbon = 3’ nucleotide.
 a 5-member, sugar ring;
 a Nucleobase;
 attached to the 1’ Carbon.
There are two major classes of Nucleotides,
 classed based upon the sugar:
 by the group, X attached to the 2’ Carbon.
 RNA contains a ribose sugar (X = OH).
 DNA contains a 2’-deoxyribose sugar (X = H).
Standard Nucleobases of
DNA
Nucleotides in DNA contain 4 types of
Nucleobases:
 2 Purines (2-ring bases):
 Adenine (A)
 Guanine (G)
 2 Pyrimidines (1-ring bases):
 Thymine (T)
 Cytosine (C)
 All are planar, and thus achiral.
 R indicates point of attachment to the 1’ C of
2’-deoxyribose.
Standard Nucleobases of RNA
Nucleotides in RNA also contain 4
Nucleobases:
 2 Purines (2-ring bases):
 Adenine (A)
 Guanine (G)
 2 Pyrimidines (1-ring bases):
 Uracil (U)
 Cytosine (C)
 Same as in DNA, except for Uracil, which replaces
Thymine.
 H substituted for Thymine’s 5’ methyl-group.
 R indicates point of attachment to the 1’ C of ribose.
Non-standard Nucleobases
Non-standard Nucleobases also exist:
 some quite common in cells.
Methylated Cytosine in DNA
 ~3%-5% of Cytosine (Human DNA).
 methylation:
 down-regulates transcription.
 protects DNA from restriction cleavage.

Transfer RNA ~10% modified bases…

 required for tRNA structure.
 Example: Pseudouridine.
 basically, “rotated” Uracil…
 5’ attached (Uracil is 1’ attached).

Here, attention restricted to standard bases.

Nucleic Acid Primary
Structure
Both DNA and RNA:
 are linear chains of nucleotides.
 linked by 5’,3’ phosphate diester
bonds.
 chain forms a negatively charged
backbone (hydrophilic).
Each chain has definite
polarity:
 two chemically distinct ends:
 5’ end (top).
 3’ end (bottom).
 by convention, oriented 5’ to 3’.
Primary Structure:
 sequence of Nucleobases, 5’ to
Polynucleotide Structure:
Overview
Structure defined by monomer torsion angles:
 many more per monomer than polypeptides:
 backbone: 6 angles (α – ζ).
 sugar ring: 5 angles (νo – ν4).
 sugar-base orientation: χ .
 nucleobase: 0 (planar).
 linked chain much more flexible.
 permissible 2o structures still helical.
Double-stranded (ds) forms:
 involve base-pairing across strands.
 similar to β-sheets.

Single-stranded (ss) forms:

 form globular 3o structures, similar to
folded polypeptides.
The Sugar Pucker
Conformation of a Nucleotide sugar ring:
 characterized by sugar pucker:
 deviation of the ring from planarity.
 defined by the position of the
C2’ and C3’ atoms;
 relative to the plane (C1’, O4’, C4’).

4 distinctive types of pucker:

 either the C2’ or C3’ atoms deviates from the plane.
 devation either above (-endo) or beneath (-exo) the
plane.
 here, ‘above’ means towards the base (‘internal’

puckering).
The Pseudorotation Angle
The torsion angles of the sugar ring (νo-ν4):
 constrained by chemical bonding.
 although not rigidly restricted, rotations are
correlated:
 variations in one requires variations in all the others.
Sugar ring torsion angles may be treated
together:
 as a single, ‘Pseudo-rotation’ angle, Ψ:

 The two major sugar conformations are defined as:

 C3’-endo for (0o <= Ψ <= 36o)
 A-Helix (RNA)
 C2’-endo for (144o <= Ψ <=190o)
Rotation about the Glycosidic
Bond
Rotations about the glycosidic bond also
constrained.
 angle of rotation denoted χ.
 rotation restricted in a base-dependent fashion:
 due to steric clash b/w base and sugar.
 large impact on structure.
Two orientations:
 anti (+180o <= χ<=+90o)
and (-90o <= χ <= -180o)
 base extended out from the sugar.
 syn (-90o <=χ<= +90o)
 base sitting above the ring.
 more compact;
 sterically disfavored.
 particulary for pyrimidines.
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Nucleic Acid 2 Structure
The 2o structures of DNA and RNA are all
helical.
 similar conceptually to polypeptides…
 with an important difference:
Nucleic Acid helices require at least 2 strands:
 either from 2 different polynucleotide chains…
 or from different regions of 1 chain.
Strands are usually H-bonded to form base-
pairs;
 may also form base triplets or quadruplets;
The best characterized Nucleic acid 2o
structures are:
 the B-helix: the standard helix of DNA.
Helix Formation in DNA
In genomic DNA, helices usually formed by 2
polymers:
 double-stranded DNA (dsDNA).
 shown conceptually, at right.
 here, helical structure omitted.

Strands oriented anti-parallel:

 5’-3’ vs. 3’-5’.
 each pair of bases aligned and H-bonded;
 Watson-Crick base pairing.
 base pairing is intermolecular.
 unit behaves as a single polymer.
 described in terms of number of base pairs.
Watson-Crick Base Pairing
Base-pairing in DNA is Watson-Crick:
 dG is paired with dC (3 H-bonds)
 dT is paired with dA (2 H-bonds)
 the 2 strands thus related by sequence:
 referred to as Watson-Crick
complementary.
Many pairs can form H-bonds…
so why these 2 base-pairs?
 points of attachment to the backbones
are equally spaced.
 allows a regular helix.
 will define a uniformly wide
major groove.
Base-pairing in RNA
Base-pairing is also important in RNA.
 however, RNA typically single-stranded.
 folding intramolecular, more varied than DNA.
 also forms well-defined helices (2o structure).
 helices aggregate into globular shapes (3o structure).
 much like polypeptides.
 each helix is a pair of H-bonded, antiparallel regions.
Base-pairing primarily Watson-Crick:
 rG paired with rC (3 H-bonds)
 rU paired with rA (2 H-bonds)
However, many other pairs common.
 ‘mismatched’ pairs of all kinds occur:
 especially GU wobble-pairs and tandem GA’s.
 A-helix much more tolerant to mismatches.
Base-pair and Base-step
Parameters

Backbone conformations of DNA, RNA double-

helices:
 related to the base-pair conformations:
 base-pair twisting, shifting, sliding,
relative to each other.
 typically described by base-pair and
base-step parameters.
Base-step Parameters ( ):
 describe the relative conformations
of 2 adjacent base-pairs.
 helical twist, roll, tilt, rise, and slide.
Base-pair Parameters ( ):
 describe the relative conformations
of 2 bases in one pair.
 Propeller Twist (ω).
The B-Helix of Watson and
Crick
The standard helix for DNA.
 right-handed, antiparallel double-helix.
 favored by high humidity conditions.
B-helix has 101 symmetry:
 motif = 1 base-pair (monomer).
 helical repeat, c = 10 base-pairs/turn.
 actually, varies from 10-10.5 bps/turn.

Parameters:
 rise, h = 0.34 nm/base-pair.
 tilt, τ = 1o (bps almost perp. to the axis).
Torsion angles:
 nucleotides in the anti conformation.
 sugars primarily 2’-endo.
B-Helix (cont.)
Two Gross Features:
 Major groove:
 this is where the bases are
exposed…
 wide and quite deep.
 involved in protein recognition.
 Minor groove:
 narrow and also quite deep.
 lined by a ‘permanent’ spine of
H20 molecules.
The B-helix not adopted by
RNA.
 due to steric hindrance:
 between each 2’-0H,
 and the adjacent 5’ phosphate.
 even a single ribonucleotide
The A-Helix
The standard helix for RNA.
 right-handed, antiparallel double-helix.
 shorter and fatter than the B-helix.
A-helix has 111 symmetry:
 motif = 1 base-pair (monomer).
 helical repeat, c = 11 base-pairs/turn.
Parameters:
 rise, h = 0.26 nm/base-pair;
 tilt, t = 19o (substantial tilt).
Torsion angles:
 nucleotides in the anti conformation.
 sugars primarily 3’-endo.
A-Helix (cont.)
Like the B-helix, has 2
grooves:
 Defined relative to the grooves
of B-DNA
 Major groove:
 narrow, but very deep.
 Minor groove:
 becomes very broad and
shallow.
May also be adopted by
DNA.
 A-form favored by:
 low humidity, alcohols and salt.
 Sequences with non-alternating
dG:dC base-pairs.
 also adopted by DNA/RNA
The Z-Helix
Can be adopted by either DNA or
RNA.
 left-handed, antiparallel double-helix.
 the narrowest of the 3 helices.
Narrowness imposes requirements:
 On Conditions:

 high salt required to minimize

repulsion b/w the two backbones.
 On Sequence:
 to fit, every other base must be syn.
 problem: syn sterically inhibited in
pyrimidines.
 thus, each strand usually an
alternating purine/pyrimidine
The Z-Helix (cont.)
Z-helix has 65 symmetry:
 motif = 2 base-pairs (dimer).
 one syn/anti and one anti/syn.
 helical repeat, c = -12 base-pairs/turn.
 but 6 rotations of the dimer yield 1 turn.

Parameters:
 rise, h = -0.38 nm/base-pair.
 tilt, τ = 9o (small).
Torsion angles:
 nucleobases alternate b/w syn and anti.
 sugars also alternate:
 2’-endo for pyrimidines (in anti);
 3’-endo for purines (in syn).
 result…backbone Zig-Zags (hence, Z-DNA).
Other Helical Structures of
DNA

DNA may adopt many other helical structures:

 virtual ‘alphabet-soup’ of DNA helices:
 A, B, C, D, H, etc… through Z.
 most have particular condition and sequence
requirements.
 some align strands in parallel.
H-DNA is triple-stranded.
 2 strands form a regular dsDNA;
 Watson-Crick base-paired.
 3rd strand sits in the major groove.
 bound by Hoogsteen base-pairing.
 Sequence Requirements (example):
 dsDNA: 1 strand poly-purine, 1 poly-pyrimidine.
 Hoogsteen strand: poly-pyrimidine
 together, this forms base-pair triplets.
 ds illustrates mirror symmetry.
Variations on B-DNA
B-DNA, itself is structurally dynamic,
 and can adopt a variety of forms…
Cruciform DNA:
 can form in sequences related
by dyad (2-fold rot.) symmetry.
 sequence at right folds to form
upper and lower arms.
 each arm forms a DNA hairpin.

A Tracts:
 sequences with the repeating motif:
 d(AAAATTTT)
Nucleic Acid Tertiary
Structure
Nucleic acids also form structures beyond
helices.
 recall that 3o structure refers to both:
 global, 3-D biopolymer structure;
 biopolymer topology.

ssRNA folds into compact 3o structures.

 such structures are globular in nature…
 fold in a manner similar to polypeptides.
dsDNA 3o structure has a different flavor:
 dominated by the B-helix.
 However, helical structures may be supercoiled:
 3o structure of DNA refers to DNA topology.
 Important for compaction into chromosomes.
 Supercoiling may also induce local, alternative
structures:
 o
Transfer RNA: 2 Structure
Transfer RNA (tRNA).
 1st nucleic acid structure determined from 1 crystal
(1974).
 Shares many features with other folded RNAs:

 provides a model for general RNA-folding.

 The ‘canonical’ tRNA molecule:
 tRNAPhe of Yeast.

Standard tRNA representation:

 as a cloverleaf.
 emphasizes 2o structure:
 4 paired regions that form A-helices.
 anticodon loop (complements mRNA).
 the amino acid attachment site (3’ end).
 o
Transfer RNA: 3 Structure
3o Structure: The relationship between the
helices and loops.
 tRNA is more compact than a cloverleaf.
 dominant feature: L-shape,
 with two perpendicular arms…
 5’ and 3’ ends terminate 1 arm.
 anticodon loop terminates the other.
 shown labeled in terms of the cloverleaf structure.
Two (not 4) distinct domains:
 one for each arm of the structure.
 domainal structure clearer on a
topological projection…
tRNA: Topological
Projection
Two distinct domains:
 First Domain (vertical):
 formed by 2 stacked A-helices:
 D-stem and Anticodon stem.
 Second Domain (horizontal):
 formed by 2 stacked A-helices:
 T-stem and Acceptor stem.
Stabilized at the elbow:
 D-loop and T-loop interact,
 forming base-triplets.
Bases in the anticodon loop are also stacked.
 single-base interaction.
Yeast tRNAPhe structure:
 provides a general model for folding of other tRNAs.
 A-helix lengths vary… but overall shape conserved.
General RNA Folding
Yeast tRNA structure also generally useful:
 provides a set of basic rules and templates.
 used extensively to model other RNA structures.
Two very simple rules:
 base stacking will be maximized,
 both within and between helices.
 base pairing also maximized.
 i.e., bases pair whenever possible.

Example: Transactivating RNA Sequence

 TAR enhances transcription of genes
encoded by the HIV virus.
 folding modeled by comparison with tRNA:
 an analogous bulged stem-loop structure.
 o
DNA 3 Structure
Unlike RNA, DNA dominated by the double-
helix.
 however, DNA still exhibits 3o structure.
 how so?…by the coiling of the helix axis.
The twist is the number of turns in a dsDNA:
 twist = Tw = N/c turns,
 N = total number of base-pairs.
 c = helical repeat.
 for relaxed, linear B-DNA, c =<c> =10.5;
 so that, Tw = N/10.5.

Ex: linear, relaxed dsDNA; length N = 147

bps:
 Twist, Tw = 147/10.5 = 14 turns.
Topology of Free, Linear
dsDNA
Consider unwinding our dsDNA by 2 turns:
 by rotation of the helix ends by –2 turns.
 Note: requires energy.
 New Twist = Tw’ = Tw + ∆Tw
= 14 – 2 = 12 turns.
 This dsDNA is now underwound:
 since Tw < (N/10.5).
 Because the ends are unrestricted, our structure still
linear.
The Topology of Circular DNA
Let’s say we now join the two ends,
 forming a closed dsDNA circle (ccDNA).
 twist remains unchanged, at Tw = 12.
 topology also described by:
The Writhe, Wr:
 number of times the helix-axis coils
around itself.
 here, Wr = 0.
The Linking Number, Lk:
 number of times each strand crosses the other.
 note Lk must be an integer.
 here, Lk = 12 turns.
Now: changes in Tw accompanied by changes in
Wr:
ccDNA Supercoiling
For instance: ∆Tw = +2 turns…
 returning the helix to Tw = 14 turns…
 Helix again B-DNA;
 but also straining the closed circle,
 causing supercoiling.
Supercoiling expressed as Writhe,
Wr.
 Two types of supercoiling:
 positive: Wr > 0 (left-handed).
 negative: Wr < 0 (right-handed).
 in our example: Wr = -2 < 0.
White’s Equation (uncut strands):
Lk = Tw + Wr
 While strands are uncut, Lk is
constant.
Supercoiling in Chromatin
Supercoiled DNA also observed in chromatin
structure:
 eukaryotic cells (linear dsDNA).
 also negatively supercoiled.
 wraps twice around nucleosome
core proteins.
 in a left-handed direction.
 c is reduced to ~10.2 bps/turn.

Analogous to supercoiling in free ccDNA.

 crossovers of free (-)-supercoiled ccDNA
right-handed, but:
 also left-handed, if wrapped around a
cylinder.
 This explains our convention…
Superhelical Strain
Superhelical strain in a ccDNA
 measured by the the superhelical density, σ =
Wr/Tw:
 the number of supercoils/helical turn:
 σ = -0.006 for both eukaryotic and prokaryotic
DNAs.
 mildly negatively supercoiled.

The standard state is the lowest energy state:

 relaxed, circular B-DNA.
 Two = N/10.5; Wro = 0; Lko = Two.
 all changes defined relative to this state:
 ∆Tw = Tw – Two
 ∆Wr = Wr – Wro
 ∆Lk= Lk – Lko
Topoisomers
The overall configuration of a ccDNA:
 is specified by Lk, the linkage number.
 also referred to as the ‘topological state’.
 different topological states: topoisomers.
Bacterial plasmids can exist in various
topoisomeric forms.
 Example - the plasmid, pBR322:
 Discrete |Wr| values resolved using Gel Electrophoresis:
 mobility increases with |Wr|.
 Lanes indicate increasing |Lk|, from
left to right (all B-DNA)…
 Lane 1 (far left): A relaxed ccDNA.
 Lk ~ N/10.5…|Wr| ~ 0.
 Lanes 2 and 3: Mixed populations
 Intermediate |Lk| values…
 Broad distribution of |Wr| values.
 Lane 4: A highly supercoiled ccDNA
Changes in Topological State
The thermodynamic partitioning of superhelical
strain:
 b/w states with the same topological configuration:
 i.e., the same Lk.
 but different conformations:
 i.e, ∆Tw and ∆Wr values…
 will be discussed in Lecture 12.
Changing the Topological state of a ccDNA:
 requires a change in Lk.
 this requires the breakage of 1 or both backbones.
Topoisomerases catalyze changes in Lk.
 they always act to relieve strain.
 changes always obey the relationship:
Conclusion
In this Lecture, we have:
 Discussed the structural characteristics of Nucleic
Acids.
 The standard helices adopted by DNA and RNA (2o
structure):
 A-Helix (standard for RNA),
 B-Helix (standard for DNA),
 Z-Helix.
 The 3o structures of Nucleic Acids:
 Folding of RNA to form globular structures.
 Supercoiling of DNA (topology).

In the next Lecture, we begin our discussion of

biopolymer structure prediction:
 With a review of Thermodynamics and the 1st Law.