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Lecture 6 Nucleic AcidStructure
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Introduction
The term, Nucleic Acid: refers to the functional forms of polynucleotides.
Nucleic acid structure:
follows many of the principles we learned forpolypeptides.
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
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Nucleotides
The monomer building blocks of Nucleic Acidsare 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).
d d l b f
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Standard Nucleobases ofDNA
Nucleotides in DNA contain 4 types ofNucleobases: 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.
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Standard Nucleobases of RNA
Nucleotides in RNA also contain 4Nucleobases: 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 replacesThymine. H substituted for Thymines 5 methyl-group.
R indicates point of attachment to the 1 C of ribose.
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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.
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Nucleic Acid PrimaryStructure
Both DNA and RNA: are linear chains of nucleotides.
linked by 5,3 phosphate diesterbonds.
chain forms a negatively chargedbackbone (hydrophilic).
Each chain has definitepolarity:
two chemically distinct ends: 5 end (top).
3 end (bottom).
by convention, oriented 5 to 3.
Primary Structure: sequence of Nucleobases, 5 to
P l l tid St t
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Polynucleotide Structure:Overview
Structure defined by monomer torsion angles: many more per monomer than polypeptides:
backbone: 6 angles ( ).
sugar ring: 5 angles (o 4).
sugar-base orientation: . nucleobase: 0 (planar).
linked chain much more flexible.
permissible 2o structures still helical.
Double-stranded (ds) forms: involve base-pairing across strands.
similar to -sheets.
Single-stranded (ss) forms:
form globular 3
o
structures, similar tofolded polypeptides.
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The Sugar Pucker
Conformation of a Nucleotide sugar ring: characterized by sugar pucker:
deviation of the ring from planarity.
defined by the position of theC2 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) theplane.
here, above means towards the base (internalpuckering).
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The Pseudorotation Angle
The torsion angles of the sugar ring (o-4): 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 treatedtogether: as a single, Pseudo-rotation angle, :
The two major sugar conformations are defined as: C3-endo for (0o
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Rotation about the GlycosidicBond
Rotations about the glycosidic bond alsoconstrained. angle of rotation denoted . rotation restricted in a base-dependent fashion:
due to steric clash b/w base and sugar. large impact on structure.
Two orientations: anti (+180o
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Nucleic Acid 2o Structure
The 2o structures of DNA and RNA are allhelical. 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 2ostructures are: the B-helix: the standard helix of DNA.
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Helix Formation in DNA
In genomic DNA, helices usually formed by 2polymers: 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.
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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-bondsso why these 2 base-pairs?
points of attachment to the backbonesare equally spaced.
allows a regular helix.
will define a uniformly widemajor groove.
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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 GAs.
A-helix much more tolerant to mismatches.
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ase-pa r an ase-s epParameters
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 ().
Th B H li f W t d
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The B-Helix of Watson andCrick
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, = 1o (bps almost perp. to the axis).
Torsion angles: nucleotides in the anti conformation. sugars primarily 2-endo.
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B-Helix (cont.)
Two Gross Features: Major groove:
this is where the bases areexposed
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 byRNA. due to steric hindrance:
between each 2-0H,
and the adjacent 5 phosphate. even a single ribonucleotide
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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.
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A-Helix (cont.)
Like the B-helix, has 2grooves: 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 byDNA. A-form favored by:
low humidity, alcohols and salt. Sequences with non-alternating
dG:dC base-pairs. also adopted by DNA/RNA
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The Z-Helix
Can be adopted by either DNA orRNA. left-handed, antiparallel double-helix. the narrowest of the 3 helices.
Narrowness imposes requirements: On Conditions:
high salt required to minimizerepulsion b/w the two backbones.
On Sequence: to fit, every other base must be syn.
problem: syn sterically inhibited inpyrimidines.
thus, each strand usually analternating purine/pyrimidine
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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, = 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). resultbackbone Zig-Zags (hence, Z-DNA).
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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.
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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
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Nucleic Acid TertiaryStructure
Nucleic acids also form structures beyondhelices. 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, alternativestructures:
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Transfer RNA: 2o 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).
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Transfer RNA: 3o Structure
3o Structure: The relationship between thehelices 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
: opo og ca
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: opo og caProjection
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.
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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.
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Topology of Free Linear
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Topology of Free, LineardsDNA
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 stilllinear.
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The Topology of Circular DNA
Lets 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 inWr:
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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.
Whites Equation (uncut strands):Lk= Tw + Wr
While strands are uncut, Lk isconstant.
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Supercoiling in Chromatin
Supercoiled DNA also observed in chromatinstructure: 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 ccDNAright-handed, but: also left-handed, if wrapped around a
cylinder. This explains our convention
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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 varioustopoisomeric 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
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Changes in Topological State
The thermodynamic partitioning of superhelicalstrain: 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:
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