Nucleic Acids

• Nucleic acid: are polymers

of Nucleotides linked with

3’, 5’- phosphodiester

bonds

• Nucleotide residues are all

oriented in the same

direction (5’ to 3’) giving

the polymer directionality.

• The sequence of DNA

molecules is always read

in the 5’ to 3’ direction

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3'

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Phospho diester bond formation

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5'

Esterification reaction between 2 nucleotides, forming the linkage

• Nucleotide monomers are joined by 3’-5’ phosphodiester linkages

to form nucleic acid (polynucleotide) polymers

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3'

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• Phosphodiester bonds can be cleaved hydrolytically by

chemicals or enzymatically by deoxyribonucleases

(DNA) or ribonucleases (RNA)

5' 3'

DNA• 1o Structure - Linear array of nucleotides and their

sequence can be determined by different methods. • 2o Structure – double helix• 3o Structure - Super-coiling, stem-loop formation• 4o Structure – Packaging into chromatin

DNA Secondary structure• DNA is double stranded with antiparallel strands• Right hand double helix• Three different helical forms (A, B and Z DNA.

Properties of DNA Double Helix

* The two chains are coiled around a common

axis

* The chains are paired in an anti-parallel

manner

* Distance between the 2 sugar-phosphate

backbones is always the same, give DNA

molecule a regular shape.

* Plane of bases are oriented perpendicular to

backbone

* Hydrophilic sugar phosphate backbone winds

around outside of helix

* Noncovalent interactions between upper and

lower surfaces of base-pairs (stacking) forms

a closely packed hydrophobic interior.

* Hydrophobic environment makes H-bonding

between bases stronger (no competition with

water)

Bases from two adjacent DNA strands can hydrogen bond•Adenine pairs with thymine using two H-bonds

•Guanine pairs with cytosine using three H-bonds

H-bonding of adjacent antiparallel DNA strands form double helix structure

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View down the Double Helix

Sugar-phosphatebackbone

Hydrophobic Interior with

base pair stacking

Structure of Structure of DNA Double DNA Double

HelixHelix• Right handed

helix• Rise = 0.33nm

nm/nucleotide• Pitch = 3.4 nm /

turn• 10.4 nucleotides

per turn• Two groves –

major and minor

* Within groves, functional groups on

the edge of base pairs exposed to

exterior

* involved in interaction with proteins.

Factors stabilizing DNA double

Helix

* Hydrophobic interactions – burying

hydrophobic purine and pyrimidine

rings in interior

* Stacking interactions – van der Waals

interactions between stacked bases.

* Hydrogen Bonding – H-bonding

between bases

* Charge-Charge Interactions –

Electrostatic repulsions of

negatively charged phosphate

groups are minimized by interaction

with cations (e.g. Mg2+)

Three major structural forms of DNA

A: right-handed, short and broad, 2.3 A, 11 bp per turn B: right-handed, longer, thinner, 3.32 A, 10 bp per turn Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn, Found in G:C-rich regions of DNA

Structural forms of the double helixStructural forms of the double helixThree major structural forms of DNAB-form, described by Watson and CrickIt is right handed helix with 10 residues per 360° turn of the helixThe plane of bases perpendicular to helical axisChromosomal DNA consists primarily of B-DNA

A-DNA form

Is produced by moderately dehydrating the B formIt is right-handed helix Contains 11 base pairs per turnThe planes of the base pairs are tilted 20 ° away from the

perpendicular to helical axis

Z-DNA form contains 12 bp per turnIs left handed helix Contains 12 about 12 base pair per turn The deoxyribose phosphate backbone forms a “Zigzag structure”

Right handed helix

Structure of DNA Double Structure of DNA Double HelixHelix

Nucleoid

-Prokaryotic organism contains a single, double

stranded, supercoiled, circular chromosome, and it is

associated with histone-like proteins and some RNA to

form a nucleoid

Plasmid

–Most species of bacteria contain small, circular,

extrachromosomal DNA molecules that called Plasmid.

–Plasmid DNA carries genetic information and undergoes

replication that may or may not related to the

chromosomal division.

–Plasmid carries genes that convey antibiotic resistance

to the host bacterium and may facilitate the transfer of

genetic information from one bacterium to another

–Plasmids used as vectors in recombinant DNA

technology

Heating up to 70 – 90C° the DNA double helix denatures,H-bonds are broken, bases unstack, and the strands separate.

Separation of the two DNA strands in the double helix

Renaturation (annealing) at

lower temperatures occurs in 2

steps.

1. Complementary bases pair.

2. The rest of the structure forms

cooperatively; it “zips-up”.

DNA with high G:C content

denatures at a higher Temp.

than A:T rich segments.

DNA sequence Determines DNA sequence Determines Melting PointMelting Point

• Double Strand DNA can be denatured by heat (get strand separation)

• Can determine degree of denturation by measuring absorbance at 260 nm.

• Conjugated double bonds in bases absorb light at 260 nm.

• Base stacking causes less absorbance.

• Increased single strandedness causes increase in absorbance

Melting Point Single strand has higher relative absorbance at 260 than dose double stranded DNA.Increasing the denaturation increases the absorbanceMelting point: the temperature at which the half of the helical structure is lost

DNA sequence Determines DNA sequence Determines Melting PointMelting Point

* Melting temperature related to G:C and A:T content.

* 3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.

DNA supercoiling:

• Supercoiling: means the

coiling of the coil.

• Typical phone cord is coiled

like a DNA helix and the

coiled cord can itself coil in a

supercoil

• A number of measurable

properties of supercoiling

have been established

DNA 3DNA 3oo Structure Structure• Supercoiling • Cruciform structures

Significance of DNA Supercoiling:

DNA SupercoilingDNA Supercoiling

•Supercoiling is a property

of circular DNA not linear

•The number of helical

turns in a linear 260-bp

DNA duplex in the B-DNA

form is 25 (260/10.4)

• Joining the two ends

Relaxed circular DNA (also

has 25 helical turn)

Another circular DNA can be formed by unwinding the linear duplex by two turns before joining its ends

This circular DNA contains 23 turns of B-helix and unwound

loop

In vivo most DNA is negatively supercoiled

A supercoiled DNA molecule is more compact than a relaxed DNA

molecule of the same length supercoiled DNA moves faster than

relaxed DNA in centrifuged

Topological parameters that describe supercoling Linking number Lk: the number of times one strand of DNA

winds around the other in the right-handed direction. Molecules

that differs only in linking number are called topological isomers

or topoisomers Number of turns of Watson-Crick helix “T” (Twisting number) Number of turns o superhelix “W” (writhing number)

L=T + W

T, W can be non-integral but L should be integral

Supercoiled DNA is favored over unwound DNA because it

contains more paired bases

Enzymes called topoisomerases or gyrases can introduce or

remove supercoils

DNA SupercoilingDNA Supercoiling

Certain DNA sequences adopt unusual structures Certain DNA sequences adopt unusual structures •Palindrome: a word that spelled identically reading forward or backward (ROTATOR). •This term is applied to DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA, such sequences are self-complementary within each strand • When inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat.

Palindromes have potential to form hairpin or Palindromes have potential to form hairpin or cruciform cruciform

Cruciform: cross-shaped structureCan form intrachain base pairing

DNA 4DNA 4oo Structure: Chromosome Structure Structure: Chromosome Structure

• In chromosomes, DNA is tightly associated with proteins

• 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

Nucleosome StructureNucleosome Structure

• Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins

• % major histone proteins: H1, H2A, H2B, H3 and H4

• Histone octamers are major part of the “protein spools”

• Nonhistone proteins are regulators of gene expression

Nucleosome StructureNucleosome Structure

• High content of Lysine and arginine (+ve charge)

•4 major histone (H2A, H2B, H3, H4) proteins for octomer

•200 base pair long DNA strand winds around the octomer

•146 base pair DNA “spacer separates individual nucleosomes

•H1 protein involved in higher-order chromatin structure.

•Chromatin looks like beads on string

Organization of Organization of Eukaryotic DNAEukaryotic DNA

Organization of Eukaryotic Organization of Eukaryotic DNADNA

The End

•4 major histone (H2A, H2B, H3, H4) proteins for octomer

•200 base pair long DNA strand winds around the octomer

•146 base pair DNA “spacer separates individual nucleosomes

•H1 protein involved in higher-order chromatin structure.

•W/O H1, Chromatin looks like beads on string