Nucleic Acids

CH339K

Monomers: Nucleotides

Component 1: 5-Carbon Sugar

Ribose vs. Deoxyribose• Difference in components of DNA and RNA• Extra hydroxyl makes RNAs much more reactive

Sugar Pucker

• Furanoses are not planar• Can pucker out of the plane of the ring at C2 or C3• Pucker effects higher order structures (or vice-versa)

Component 2: Nitrogenous Base

Purines

Pyrimidines• Cytosine and Thymine in DNA• Cytosine and Uracil in RNA

Component 3: Phosphate

Nomenclature

Names of Base Derivatives

Base Nucleoside 5'-Nucleotide

Adenine (Deoxy)Adenosine (Deoxy)Adenosine-5'-monophosphate

Guanine (Deoxy)Guanosine (Deoxy)Guanosine-5'-monophosphate

Cytosine (Deoxy)Cytidine (Deoxy)Cytidine-5'-monophosphate

Thymine (Deoxy)Thymidine (Deoxy)Thymidine-5'-monophosphate

Uracil (Deoxy)Uridine (Deoxy)Uridine-5’-monophosphate

Syn and Anti Conformations

Syn / anti energetics

From: Neidle, S. (2008) Principle of Nucleic Acid Structure Elsevier, London, pg. 33

Condensation – Polymer Formation

Phosphodiester Linkages

Simple Condensation is Energetically Unfavorable

Go‘≈ +25 kJ/mol Keq=4.15*10-5

Synthesis is from the triphosphate

Energetics:

Phosphodiester formation - +25 kJ/molnTP cleavage – -31 kJ/molPyrophosphate cleavage - -19 kJ/mol

Keq = 24100

Tautomeric Forms of Bases-NHx groups can be in the amino or imino conformation

=O groups can be in the keto or enol conformation

The predominant form for the free base is not necessarily the predominant form in the nucleotide

Lack of basic O-Chem knowledge caused problems for Watson and Crick when they were trying to figure out the structure of DNA

Keto Enol

Base Pairing

keto

keto

amino

amino

Cytosine Guanine

Animation

Secondary Structure of Nucleic Acids

• Helical• Result of base

pairing• Defined by

– Pitch

– Rise

– In turn governed by structure of the monomers

B Helix  B form

Helical Sense Right handed

Diameter ~20Å

Base pairs per helical turn 10

Helical twist per base pair 36°

Helix pitch (rise per turn) 34 Å

Helix rise per base pair 3.4 Å

Base tilt normal to the axis 6°

Major groove Wide & deep

Minor grooveNarrow &

deep

Sugar pucker C2'-endo

Glycosidic bond Anti

Typical DNA

Determination of helix parameters

Rosalind Franklin’s Diffraction Photo of B-DNA

A Helix

  A form

Helical Sense Right handed

Diameter ~26 Å

Base pairs per helical turn 11

Helical twist per base pair 33°

Helix pitch (rise per turn) 28 Å

Helix rise per base pair 2.6 Å

Base tilt normal to the axis 20°

Major groove Narrow & deep

Minor groove Wide & shallow

Sugar pucker C3'-endo

Glycosidic bond Anti

RNA, DNA/RNA hybrids, dehydrated DNA

Z Helix  Z form

Helical Sense Left handed

Diameter ~18 Å

Base pairs per helical turn 12 (6 dimers)

Helical twist per base pair 60° (per dimer)

Helix pitch (rise per turn) 45 Å

Helix rise per base pair 3.7 Å

Base tilt normal to the axis 7°

Major groove Flat

Minor groove Narrow & deep

Sugar puckerC2'-endo (pyrimidines)

C3'-endo (purines)

Glycosidic bondAnti (pyrimidines)

Syn (purines)Alternating Purine-Pyrimidine

Z DNA Function?

• Z DNA is antigenic• Antibodies are found in autoimmune disorders

like systemic lupus erythematosus• Antibodies bind to puffs in Drosophila polytene

chromosomes• Also bind macronuclei of ciliates• Z DNA-prone sequences found in transcription

start sites• May act as spacer between RNA polymerases• Z DNA binding proteins required for

pathogenicity by vaccinia and smallpox

Helix Parameters Summarized  A form B form Z form

Helical Sense Right handed Right handed Left handed

Diameter ~26 Å ~20Å ~18 Å

Base pairs per helical turn 11 10 12 (6 dimers)

Helical twist per base pair 33° 36° 60° (per dimer)

Helix pitch (rise per turn) 28 Å 34 Å 45 Å

Helix rise per base pair 2.6 Å 3.4 Å 3.7 Å

Base tilt normal to the axis 20° 6° 7°

Major groove Narrow & deep Wide & deep Flat

Minor groove Wide & shallow Narrow & deep Narrow & deep

Sugar pucker C3'-endo C2'-endoC2'-endo (pyrimidines)

C3'-endo (purines)

Glycosidic bond Anti AntiAnti (pyrimidines)

Syn (purines)

Major and Minor Grooves

Grooves provide access to base sequence

• Telomere binding protein

• -helix fits into major groove

• Side chains can recognize bases

Another Example

cro Repressor protein of bacteriophage .

Small (66 amino acids)

Forms dimers

Binds to specific sites on DNA that activate / deactivate genes

Expression of cro results in the phage entering the lytic cycle

Ab

sorp

tio

n o

f U

V L

igh

t

UV Absorption Spectrophotometry

Beer-Lambert Law

clTA

I

IT cl

o

log :Absorbance

elyalternativor

10 :nceTransmitta

c = concentrationc = concentration

l = path lengthl = path length

= extinction coefficient= extinction coefficient

An Absorbance = 2 means that only 1% of the incident beam is getting through. An Absorbance = 2 means that only 1% of the incident beam is getting through.

Transmittance and Absorbance

Absorbance vs. Concentration Transmittance vs. Concentration

Physical Properties - Absorbance

Physical Properties - Hypochromicity

• Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases, an effect called hypochromism

• Rules of thumb:– 280 dsDNA: 20

– 280 ssDNA/RNA: 37.5

– 280 small oligonucleotides: 50

1) Calculated spectrum of equivalent mixture of free nucleotides

2) Double stranded RNA (38% G+C)

3) Single stranded RNA (38% G+C)

From Cox, R. A. (1970) Conformation of Nucleic Acids and the Analysis of the Hypochromic Effect, Biochem. J. (1970) 120, 539-547

Denaturation: “Melting”

• Heat, alkali cause the double helix to unwind

• As H-bonds break, they form “bubbles” in the helix

• As the equilibrium shifts towards H-bonds breaking, the bubbles coalesce

• The strands come apart

As temperature increases, local denatured regions coalesce

Effect of G+C content on Tm

ss Bubbles Coalesce until Strands Separate

Effects of changing o’ and So’

Artificially generated curves

DNA Sequencing – Sanger Method

DNA Sequencing - Sequencers

Polymerase Chain Reaction(aka DNA Amplification)

Internal Structure

Palindromes and inverted repeats tend to be sites for recognition by proteins

Palindromes:

Kay, a red nude, peeped under a yak

Some men interpret nine memos

Campus Motto: Bottoms up, Mac

Internal Structure (cont.)

Replication Origin of Duck Hepatitis B

Nonstandard Base Pairs

Triplex DNA Structure

A) Duplex DNA Structure

B) Triplex DNA with 3rd Strand in Major Groove

Bissler, John J. (2007) Triplex DNA and human disease, Front. Biosci. 12: 4536-4546.

Duplex, Triplex, and Quadraplex

Quadraplexes are found in telomeres

Telomeres contain repeats of d(GGTTAG), which form quadraplexes.

Nucleic acids can form higher – order three dimensional structures…

…and it’s a good thing.

Tertiary Structures - tRNAs

tRNAs can contain a variety of modified nucleotide bases

Tertiary Structure - Viroids

• Viroids are small, naked circular, mostly double-stranded RNAs which infect plants

• Host RNA Polymerase copies the RNA many times

• Self-cleavage into individual lengths• Host ligases close into circles

Potato Spindle Tuber Viroid

African oil palm with cadang-cadang like viroid disease

Frequency of Cadang-Cadang in Coconut palms from two Phillippine provinces 1951-1976

From Zelazny, B., and Pacumbaba, E. (1982) Plant Disease 66: 547-549.

Tertiary Structures (cont.)

Examples of some specialized RNAs

E. coli 16S ribosomal RNA

Nucleases

• Nucleic acids can be hydrolyzed enzymatically by nucleases;

• Nucleases belong to the class of phosphodiesterases;– Cleavage at the 3’ side by “a” type nucleases

(leaves 5’ phosphate);– Cleavage at the 5’ side by “b” type nucleases

(leaves 3’ phosphate);– Endonucleases cleave in the middle of the NA;– Exonucleases cleave from the ends.

• DNases act on DNA; RNases act on RNA.

Examples

5’ p-A-p-G-p-G-p-T-p-C-p-C-p-T-p-A-OH 3’

a-type 3’ exob-type endo

b-type 5’ exo

Word of the Day: Processivity - The ability of an enzyme to repetitively continue its catalytic function without dissociating from its substrate.

(The exonuclease examples above are not processive)

Examples

Enzyme Substrate Type

Pancreatic RNase RNA b-type (5’) endo

Snake Venom Phosphodiesterase RNA / DNA a-type (3’) exo

Spleen Nuclease RNA / DNA b-type (5’)exo

Examples

From Smith, C., and Wood, E. J. (1991) Biological Molecules, Springer, New York, pg 188

Restriction Systems - Bacteriophage

Bacteriophage T4

Restriction Systems - Bacteriophage

RestrictionEndonucleases

E. coli R

Infectivity~1

Infectivity~1 x 10-4

E. coli K

Infectivity~1

Infectivity~1 x 10-4

Kablooey!

Kablooey!

• Phage hatched from the R strain reinfect the R strain easily.

• Phage hatched from the K strain reinfect the K strain easily.

• Phage from the R strain are restricted on K

• Phage from the K strain are restricted on R.

Restriction due to endonuclease / methylase system

The endonuclease and the methylase recognize the same sequence

The endonuclease will not cut the methylated DNA

Host can discriminate its own DNA from that of a virus if the virus is raised in a bug with a different restriction system

Protective Role of Restriction Systems

Example of a restriction modification system

EcoR1 (first restriction system from E. coli strain R) recognizes a 6-base palindrome:

5'-GAATTC-3'3'-CTTAAG-5'

The methylase puts a methyl group on the underlined adenosines if the sequence is not methylated. The nuclease clips each strand between the 5' G and A of the unmethylated recognition site

5'-G AATTC-3'3'-CTTAA G-5'

The resulting overhangs are "sticky ends" - they will base pair with a complementary sequence.

Cloning using Restriction Endonucleases

There are a zillion REs for just about any palindrome

(Enzymes for 234 recognition sites available from New England Biolabs as of March 2010)

DNA Modifying Agents as Drugs

Intercalating Agents

• Stack between base pairs

• Ethidium is used as a fluorescent DNA stain

• Acridine is also used as a stain for DNA (green) and RNA (red)

• Actinomycin inhibits transcription by binding at the start site

Ethidium Bromide Intercalated into DNA

DNA stained with ethidium bromide

Reactivity of Ribonucleic Acid Due to the 2’-Hydroxyl

Base1

O

OHO

HHHH

PO

O

O

O- Base2

O

OH

HHHH

O

PO

-O

O-

Base1

O

OO

HHHH

PO

O

O

O- Base2

O

OH

HHHH

O

PO

-O

O-

base

Base1

O

OO

HHHH

PO

O

Base2

O

OH

HHHH

O

PO

-O

HO

O-

O-

H2O

Base1

O

OHO

HHHH

PO

O

Base2

O

OH

HHHH

O

PO

-O

HO

O-

O-O-

Supercoiling

• DNA in “relaxed” state - 10.4 bp/turn• If DNA is twisted, the strands become more tightly or

more loosely wound: supercoiling– in direction of helix = “positive supercoiling”

– in the opposite direction = “negative supercoiling”

• In nature, most DNA has a slight negative supercoiling that is introduced by enzymes called topoisomerases (counteract effect of transcription and replication)

Supercoiling

• Can open helix• Overwind or underwind• Changes Linking Nbr• L = T + W• Underwound DNA is

primed to uncoil– Transcription

– Replication

– Recombination

– Z-DNA formation

Linking Numbers

Negative and Positive Supercoils

Supercoiling in a viral DNA

Different levels of supercoiling in Simian Virus 40 (SV40) DNA.. SV40 may (or may not) be involved in causing human tumors. Those of us inoculated for polio prior to 1962 were probably exposed to SV40 as a contaminant of the polio vaccine.

Supercoiling of Constrained Linear DNA

Supercoiling: Energetic Considerations

• Because there are ~10.4 bp/turn in B-helical DNA, the relaxed Linking Number is

Lo = bp / 10.4

• Upon supercoiling, the change in L is

ΔL = L − Lo

• We can define superhelical density as

σ = ΔL / Lo

• The free energy involved in supercoiling is related to

ΔG / N = KRTσ2 (usually shown as ΔG = KNRTσ2)

where N = number of (constrained) base pairs and

K depends on the solution (ionic strength, concentration, etc.)

Supercoiling (cont.)

• Wrapping of DNA around nucleosome requires ≈ -0.05

Type 1 TopoisomerasesCut 1 strand of the DNAChange L by 1Involved in protein synthesis control

Type 2 Topoisomerases

Cut both strands simultaneouslyChange L by 2Most well known is DNA GyraseIn presence of ATP can induce supercoilingUnwinds DNA ahead of replication fork

Topoisomerase Action

Simian virus 40 (SV40) DNA incubated w/ a human topoisomerase for 0, 1, 3, 6, 10, and 30 minutes, going from an average of 25 superhelical turns to 0 (relaxed)

Riddle me this, Doc…

…why does DNA use Thymine instead of uracil? Seems like a waste of complexity.

Spontaneous Deamination

Asn Pro Gly CysAAT CCT GGC TGTTTA GGA CCG ACA

Asn Ser Gly CysAAT TCT GGC TGTTTA AGA CCG ACA

Frequency: 100 – 500 times per human cell per day.

That’s about 1 - 5 * 1015 per person per day.

DNA Mismatch Repair

DNA has a system to recognize uradines in the DNA strand

Glycosylase clips of the uracil base

An endonuclease clips out the sugar phosphate

Polymerase fills the gap

Expectations• Know structures of nucleotides and components.• Understand the linear, directional, backbone / base

structure of the polymer.• Understand base pairing.• Understand the properties of the helix types and what

types of nucleic acids assume which forms.• Beer’s law• Difference between primary, secondary, and tertiary

structure.• Different types of nucleases; what are restriction

systems?• Meaning and significance of supercoiling;

topoisomerases.• What the heck is spontaneous deamination?