lecture 6' - nucleic acid structure

8/14/2019 Lecture 6' - Nucleic Acid Structure

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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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lecture 6' - nucleic acid structure

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