Lecture 1 - Nucleic Acids - Su 15

7/25/2019 Lecture 1 - Nucleic Acids - Su 15

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MCB 102 - Survey of the

Principles of Biochemistry and

Molecular Biology

Lecture


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Functions of Nucleotides and

Nucleic Acids Nucleotide Functions:

!

Energy for metabolism (ATP)

! Enzyme cofactors (NAD+)

!

Signal transduction (cAMP)

Nucleic Acid Functions:! Storage of genetic info (DNA)

!

Transmission of genetic info (mRNA)

! Processing of genetic information (ribozymes)

!

Protein synthesis (tRNA and rRNA)


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!

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Nucleotides and Nucleosides


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Pyrimidine Bases

!

#$%&'()*(' ,&-). () /&%0 123 4). 523

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! 9:4;(8(' ,&-). &)8$ () 523

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Pentose in Nucleotides

!

!>1>:(/&,-:4)&'* () 523

! !>C>.*&F$>1>:(/&,-:4)&'* () 123

! 1(G*:*)% ?-;H*:*. ;&),&:74I&)' JKL

&, %0* '-


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Conformation around N-Glycosidic

Bond Relatively free rotation can occur aroundthe N-glycosidic bond in free nucleotides

There is free rotation around 6 bonds,with restrictions only around bond 4.

Conformation can be Syn or Anti.

Anti conformation is found in normal B-DNA .


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Phosphate Group

Negatively charged at neutral pH

Typically attached to 5position! Nucleic acids are built using 5-triphosphates

! ATP, GTP, TTP, CTP

!

Nucleic acids contain one phosphate moiety per nucleotide May be attached to other positions


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Nomenclature


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6&" /%%( ,& 7/&8 0,-"#,"-%09 /2:%09 2/( 0;:1&$0

%-


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6&" /%%( ,& 7/&8 0,-"#,"-%09 /2:%09 2/( 0;:1&$0

%- 2/( ,4-%%=$%>%- #&(%0@

Ribonucleotides


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UV Absorption Spectrum

The absorptionspectrum is independent

of ribose or deoxyribosein free nucleotides


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Minor Nucleosides in DNA

!

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S?(


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Minor Nucleosides in DNA


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Minor Nucleosides in RNA

Inosine sometimes found in the wobble positionof theanticodon in tRNA

!

Made by de-aminating adenosine

! Provides richer genetic code

Pseudouridine (!) found widely in tRNA and rRNA

! More common in eukaryotes but found also ineubacteria

!

Made from uridine by enzymatic isomerization afterRNA synthesis

! May stabilize the structure of tRNA

! May help in folding of rRNA


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Minor Nucleosides in RNA


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Polynucleotides

Covalent bonds formed via phosphodiester linkages (5

-P to 3-OH)! negatively charged backbone

Backbone is fairlyhydrophilic as the sugarresidues can form hydrogen

bonds with water.

Phosphate groups have pKanear 0, so they are completelyionized at pH 7.

The negative charges of thebackbone are neutralized byionic interactions with positivecharges in proteins, metal ionsand polyamines.


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Polynucleotides

The DNA backbone isstable.

The RNA backbone ismore unstable and can be

hydrolyzed at alkaliconditions (due to theextra OH)

Polynucleotides are linearpolymers, with no

branching or side chains.

They have directionality,and we read the sequencefrom the 5to 3.


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Hydrogen-Bonding Interactions

Two bases can hydrogen bond toform a base pair.

For monomers, large number ofbase pairs is possible.

In polynucleotide, only fewpossibilities exist

Watson-Crick base pairspredominate in double-stranded

DNA

Purine pairs with pyrimidine! A pairs with T (2Hydrogen Bonds)

! C pairs with G (3Hydrogen Bonds)


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DNA is a double helix with

antiparallel polynucleotide strands! 60* '*]-*);* (' :*4. ,:&7 D%& ^M

! 60* %U& ;04()' .(G*: () '*]-*);*M

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60* %U& ;04()' 4:* ;&7?8*7*)%4:$M

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G

"C

Z%4/(8(%$ .-* 7&'%8$ %& %0* '%4;H()< &,%0* /4'*'


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Double Helical DNA strength in

numbers Hydrogen-bonds between bases areonly slightly stronger than betweena base and H2O.

The packing of DNA bases doesn

tallow water molecules inside thedouble helix.

In the absence of H2O betweenbases >12 complementary base pairsare sufficient to hold two DNAstrands together.

The cooperativity of the manynoncovalentlinks of a zipper are a

familiar model.


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Why dont the bases usually

swivel around? Vertical sharing ofaromatic delocalized p-electrons between basesgenerates stacking bonds

(Zusman slide).

This vertical stackingprevents the DNA bases

from rotating freelyaway from the basepairing with thecomplementary strand.

(pi-stacking)

P-electron bonding issimilar to metallic bonds,in which numerous atoms

share electrons.

+

-

+

-


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Further evidence for vertical

stacking: Aromatic dyes intercalate

between bases of DNAAromatic dyes like ethidium,

actinomycin intercalate,

stackin DNA: The resulting fluorescence is

evidence for vertical bondingby shared electrons(Lehninger p960).


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Intercalation of dyes in DNA

generates fluorescence in UV light

To a first approximation

Aromatic dyes, when theyabsorb a photon can release

energy either as:!

Thermal collision with the solution.

!

Fluorescence

Stacked in DNA pairs, it is

dehydrated, and has lessfreedom of motion and is thusless likely to release energythrough thermal collision andmore likely to release a photon.


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DNA Fluoresces in Eth-Br buffer

(Agar gel electrophoresis)


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Bases can Flip Out of the

Double Helix

Sometimes individual bases canprotrude from the double helix,a phenomenon known asbaseflipping.

This is important in DNAmethylation and DNA repair.! In fact, it is thought that

enzymes involved inhomologous recombination and

DNA repair scan DNA byflipping out one base afteranother.


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Major and Minor Grooves A result of the geometry of base

pairs.

The edges of each base pair are

exposed in the major and minorgrooves, creating a pattern of H-

bond donors and acceptors andof van der Waals surfaces thatcan be recognized. A= acceptor D= donor H= non-polar

hydrogensM= methylgroups

Major groove is extremely sensitive to the identity of the basepairs inside.

AT!A D A M (TA"M A D A)

GC!A A D H (CG"H D A A)

Minor groove has similar hydrogen bonding patterns regardlessof the base pair.

AT!A H A (TA"A H A)

GC!A D A (CG"A D A)


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Possible DNA Structures

DNA can exist in a variety ofstructures as there is asignificant number of bondswith free rotation.

All conformations contain thefollowing properties, firstdefined by Watson and Crick:

! Strand complementarity

!

Antiparallel strands! A= T and G"C base pairs.


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Possible DNA Structures

Most Common Form

A form ismore

favorablefor DNA-

RNA orRNA-RNA


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B-DNA

Right handed

Diameter ~20A

10.5 base pairs per helicalturn.

Helix rise per base pair3.4A

Almost planar baseorientation. They stacktogether closely,

excluding water. Major and minor grooves.

36A is a complete turn.

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Lecture 1 - Nucleic Acids - Su 15