Molicular Biology

Types of Molecules

• DNA: code for genetic information

• RNA– mRNA intermediates in protein synthesis– rRNA carry out protein synthesis– tRNA translate info on mRNA into a.a.

• Gene

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Nucleotide Components

• Bases + sugar + phosphate• Bases

– purine• adenine

• guanine

– pyrimidines• thymine

• cytosine

• uracilwww.freelivedoctor.com

Bases

N N

NN

NH2

N N

NN

O

NH2

N

N

O

O

CH3

N

N

O

NH2

N

N

O

O

Adenine (A)

Guanine (G)

Purines Pyrimidines

Thymine (T) (DNA only)

Cytosine (C)

Uracil (U) (RNA only)


Nucleotide Components

• Sugars– ribose

– Deoxyribose

– Rings numbered using primes

• C1 = 1’ etc

• PO43-

OH

OHOH

OHOH2C

OH

HOH

OHOH2C

ribose (RNA)

2-deoxyribose (DNA)


Nucleotide Structure

• Base-sugar-phosphate

• Base attached to C1 of sugar

• Phosphate attached to C5 of sugar

• Usually nucleotides are triphosphates, but can be mono or di

OH P O

O

O

P OO

O

P O

O

O

N

O

N

N

OH

OH

N

NH2

OH P O

O

O

P OO

O

P O

O

O OH

OH

N

N

O

O

CH3

Adenosine Triphosphate (ATP) (a ribonucleotide)

Thymidine Triphosphate (TTP) (a deoxyribonucleotide)


Nucleic Acid, a polymer of nucleotides


Nucleic Acids

• Polymer of nucleotides

• Phosphodiester linkage – 3’ of one sugar to 5’ of other– vectorial

• Sequence read 5’ - 3’

• Previous slide is C-A-A

• Next slide (a cartoon drawing) is A-T-G-C


Phosphodiester Bond


Chain elongation

• 3’ hydroxyl on chain has unshared pair on oxygen– attacks phosphorus on first phosphate of new nucleotide– Nucleophilic attack

• Driven by hydrolysis of PPi

OH P O

O

O

P OHO

O

O P OH

O

O

pyrophosphate

2

phosphate


Chain elongation

H

OO

N N

NN

O

N

P

O

O

OR

OH

OH P O

O

O

P OO

O

P O

O

O

N

O

N

N

H

OH

N

NH2

H

OO N N

NN

O

N

P

O

O

OR

O

PO

O

O N

ON

N

H

OH

N

NH2OH P O

O

O

P OHO

O

+

chain

nucleotide

3- OH

+

longer chainpyrophosphate


Base pairs

– Bases are planar• sp2 hybridization• Bases nonpolar

– In Nucleic acids, there is base pairing– Complementarity

• Purines always base-pair with pyrimidines– Cytosine hydrogen bonds with guanine– Thymine hydrogen bonds with adenine or uracil

• Due to distancing and proximity of unshared pairs and hydrogens


GC base pair

NN

N

NH

O

N HR

H

Guanine

NN

O

N

H

H

R

-- -- --

-- -- --

-- -- --

cytosine

3 H- bonds


AT base Pair

N

N

N

N

N H

H

R

adenine

NNH

O

O

R

-- -- --

-- -- -- --

thymine

2 H-bonds


A-U base Pair (RNA)

N

N

N

N

N H

RNN

O

O

H R

H

adenine uracil

2 H bonds


Base Pairs (Cartoon drawing)


DNA structure

• Double helix


DNA structure

• B-DNA is most common• Antiparallel• bases inside

– Hydrophobic– Perpendicular to helix

• stands complementary– Very important for information transfer– Each strand a template for the other.

• right handed• major and minor groove


Other forms of DNA

• Z - DNA left handed; structure not fully understood

• A- DNA; tighter helix


DNA TypesB- DNA (most common)


A-DNA (supercoiled)


Z-DNA


Look down Axis


Sequence dependent structures

• palindromes

• hairpins

• inverted repeats

• mirror repeats


RNA

• codes for protein

• no consistent secondary structure

• single stranded

• Ribose instead of deoxyribose

• Thymine (T) replaced by Uracil


RNA

O

OHOH

CH2OO P

OO

O

N

OH

CH2O

O P

OO

ON

OH

CH2OO P

OO

O P O

O

O

O N

OH

N

NN

NH2

CH2

N

O

O

N

NN

O

NH2

N

N

O

NH2

5' end

A

U

G

C

3 ' end


Transfer RNA


Instability of RNA

• RNA unstable due to reactivity of 2’ hydroxyl on ribose

• Very labile

• DNA extremely stable


Nucleic Acid Chemistry

• Denaturation– unzip by heating– reannealing– CoT curves: more GC=higher temp

• Uses of DNA to find sequences– probes– forensic


DNA probes

• Take DNA from crime scene

• DNA from suspect, random other person etc

• See what matches



• Mutations– changes in

sequence/structure

– nonenzymatic changes• thymine dimers

• deamination

• oxidative damage



• Sequencing

• PCR

• Southern Blotting

• Probes


DNA sequencing, Sanger dideoxy method

• 4 samples• Each gets template and nucleotides

– One dideoxy nucleotide– Has no 3’ OH so chain can’t elongate

• Sample 1– ddTTP…stops at T

• Sample 2– ddATP…stops at A

• Sample 3– ddCTP…stops at C

• Sample 4– ddGTP…stops at G

• Run on Gel– Read from bottom up


Sample Sanger Gel


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