2828

28-1

Organic Organic Chemistry Chemistry

William H. Brown &William H. Brown &

Christopher S. FooteChristopher S. Foote

2828

28-2

NucleicNucleicAcidsAcids

Chapter 28Chapter 28

2828

28-3

Nucleic AcidsNucleic Acids Nucleic acid:Nucleic acid: a biopolymer containing three

types of monomer units• heterocyclic aromatic amine bases derived from

purine and pyrimidine• the monosaccharides D-ribose or 2-deoxy-D-ribose• phosphoric acid

Following are names and one-letter abbreviations for the heterocyclic aromatic amine bases most common to nucleic acids

2828

28-4

Purine/Pyrimidine BasesPurine/Pyrimidine Bases

HN

N

O

O

H

N

N

NH2

O

H

HN

N

O

O

H

CH3N

N

HN

N N

N

O

HH2N

N

N N

N

NH2

H

N

N N

N

H

Uracil (U) Thymine (T) Cytosine (C)Pyrimidine

1

2

34

5

6

Guanine (G)Adenine (A)Purine

1

2

34

56 7

8

9

2828

28-5

NucleosidesNucleosides Nucleoside:Nucleoside: a building block of nucleic acids,

consisting of D-ribose or 2-deoxy-D-ribose bonded to a heterocyclic aromatic amine base by a -glycosidic bond

anomericcarbon

a - -N glycosidicbond

HH

H

H

OHOCH2

HO OH

O

O

HN

N

Uridine

β-D-riboside

uracil

1'

2'3'

4'

5'

1

2828

28-6

NucleotidesNucleotides Nucleotide:Nucleotide: a nucleoside in which a molecule of

phosphoric acid is esterified with an -OH of the monosaccharide,

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OP

O-

O-O

5'

Adenosine 5'-monophosphate(AMP)

2828

28-7

NucleotidesNucleotidesExample 28.1Example 28.1 identify these nucleotides

NO

N

O

HH

H

H

HOH

-O-P-O-P-O-CH2

NH2

(b)(a)-O -O

OO

O

HH

H

H

OH

HOCH2

HN

N N

NO

H2N

PO-

-O O

2828

28-8

Acyclovir & AZTAcyclovir & AZT

Azidothymidine (AZT) Acyclovir (drawn to show itsstructural relationship to 2-deoxyguanosine

O

O

N

HN

HOC

HN3

H

H

HH

O

CH3

O

H HHH

HOC

H2N

HN

N

O

N

N

H2H2

2828

28-9

DNA - 1° StructureDNA - 1° Structure Deoxyribonucleic acids (DNA)

• a backbone of alternating units of 2-deoxy-D-ribose and phosphate in which the 3’-OH of one 2-deoxy-D-ribose is joined by a phosphodiester bond to the 5’-OH of another 2-deoxy-D-ribose unit

Primary Structure:Primary Structure: the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule) • read from the 5’ end to the 3’ end

2828

28-10

DNA - 1° StructureDNA - 1° Structure

NO

HN

-O-P-O-CH2

O

HH

OH

HH

H

OCH3

O=P O CH2

O

-O

O-

NH

NN

N

O

NH2

O

HH

O

HH

H

5'

3'

phosphorylated5' end

free 3' end

2828

28-11

DNA - 2° StructureDNA - 2° Structure Secondary structure:Secondary structure: the ordered arrangement

of nucleic acid strands The double helix model of DNA 2° structure was

proposed by James Watson and Francis Crick in 1953

Double helix:Double helix: a type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis

2828

28-12

T-A Base PairingT-A Base Pairing A major factor stabilizing the double helix

2828

28-13

C-G Base PairingC-G Base Pairing

2828

28-14

Forms of DNAForms of DNA B-DNA

• the predominant form in dilute aqueous solution• a right-handed helix• 2000 pm thick with 3400 pm per ten base pairs• minor groove of 1200pm and major groove of 2200 pm

A-DNA• a right-handed helix, but thicker than B-DNA• 2900 pm per 10 base pairs

Z-DNA• a left-handed double helix

2828

28-15

DNA - 3° StructureDNA - 3° Structure Tertiary structure:Tertiary structure: the three-dimensional

arrangement of all atoms of nucleic acid, commonly referred as supercoiling

Circular DNA:Circular DNA: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds (Fig 28.10)

Histone:Histone: a protein, particularly rich in the basic amino acids lysine and arginine, that is found associated with DNA molecules

2828

28-16

DNA - 3° StructureDNA - 3° Structure Chromatin:Chromatin: consists of DNA molecules wound

around particles of histones in a beadlike structure• further coiling produces the dense chromatin found in

nuclei of plant and animal cells

2828

28-17

Ribonucleic Acids (RNA)Ribonucleic Acids (RNA) RNA are similar to DNA in that they, too, consist

of long, unbranched chains of nucleotides joined by phosphodiester groups between the 3’-OH of one pentose and the 5’-OH of the next. However,• the pentose unit in RNA is -D-ribose rather than -

2-deoxy-D-ribose• the pyrimidine bases in RNA are uracil and cytosine

rather than thymine and cytosine• RNA is single stranded rather than double stranded

2828

28-18

RNARNA RNA molecules are classified according to their

structure and function

Ribosomal RNA (rRNA):Ribosomal RNA (rRNA): a ribonucleic acid found in ribosomes, the site of protein synthesis

Molecular WeightRange (g/mol)

Number ofNucleotides

Percentageof Cell RNA

mRNA 25,000 - 1,000,000 75 - 3,000 2tRNA 23,000 - 30,000 73 - 94 16rRNA 35,000 - 1,100,000 120 - 2904 82

Type

2828

28-19

RNARNA Transfer RNA (tRNA):Transfer RNA (tRNA): a ribonucleic acid that

carries a specific amino acid to the site of protein synthesis on ribosomes

OBase

OHO

CH

RNH3

+

tRNA-O-P-O-CH2

amino acid, boundas an ester to itsspecific tRNA

HH H

H

C=O

O

O-

2828

28-20

RNARNA Messenger RNA (mRNA):Messenger RNA (mRNA): a ribonucleic acid that

carries coded genetic information from DNA to the ribosomes for the synthesis of proteins• present in cells in relatively small amounts and very

short-lived• single stranded• their synthesis is directed by information encoded on

DNA• a complementary strand of mRNA is synthesized

along one strand of an unwound DNA, starting from the 3’ end

2828

28-21

RNARNA• the synthesis of mRNA from DNA is called

transcription

3'-A-G-C-C-A-T-G-T-G-A-C-C-5'

5'-U-C-G-G-U-A-C-A-C-U-G-G-3'

DNA template

mRNA

2828

28-22

UUU PheUUC PheUUA LeuUUG Leu

UCU SerUCC SerUCA SerUCG Ser

UAU TyrUAC TyrUAA StopUAG Stop

UGU CysUGC CysUGA StopUGG Trp

GUCAG

CUU LeuCUC LeuCUA LeuCUG Leu

CCU ProCCC ProCCA ProCCG Pro

CAU HisCAC HisCAA GlnCAG Gln

CGU ArgCGC ArgCGA ArgCGG Arg

UCAG

G

AUU IleAUC IleAUA IleAUG* Met

ACU ThrACC ThrACA ThrACG Thr

AAU AsnAAC AsnAAA LysAAG Lys

AGU SerAGC SerAGA ArgAGG Arg

UCAG

UCAG

GUU ValGUC ValGUA ValGUG Val

GCU AlaGCC AlaGCA AlaGCG Ala

GAU AspGAC AspGAA GluGAG Glu

GGU GlyGGC GlyGGA GlyGGG Gly

*AUG also serves as the principal initiation codon.

U

U C A

C

A

The Genetic CodeThe Genetic Code

2828

28-23

The Genetic CodeThe Genetic Code Properties of the Code

• only 61 triplets code for amino acids; the remaining 3 (UAA, UAG, and UGA) signal chain termination

• the code is degenerate, which means that several amino acids are coded for by more than one triplet. Leu, Ser, and Arg, for example, are each coded for by six triplets

• for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU

• there is no ambiguity in the code; each triplet codes for one and only one amino acid

2828

28-24

Sequencing DNASequencing DNA Restriction endonuclease:Restriction endonuclease: an enzyme that

catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand• over 1000 endonucleases have been isolated and their

specificities determined• typically they recognize a set sequence of nucleotides

and cleave the DNA at or near that particular sequence

• EcoRI from E. coli, for example, cleaves as shown

5' G-A-A-T-T-C---3'EcoRI

5' G + 5'-A-A-T-T-C---3'

cleavage here

2828

28-25

Sequencing DNASequencing DNA• following are several more examples of

endonucleases and their specificities

AluI AG CT

BalI TGG CCA

FnuDII CG CG

HeaIII GG CC

Not I GC GGCCGC

Mbol GATC

SacI GAGCT C

Enzyme EnzymeRecognitionSequence

RecognitionSequence

HpaII C CGG

2828

28-26

Sequencing DNASequencing DNA Polyacrylamide gel electrophoresis:Polyacrylamide gel electrophoresis: a technique

so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide• Maxam-Gilbert method:Maxam-Gilbert method: a method developed by Allan

Maxam and Walter Gilbert; depends on base-specific chemical cleavage

• Dideoxy chain termination method:Dideoxy chain termination method: developed by Frederick Sanger

• Gilbert and Sanger shared the 1980 Nobel prize for biochemistry for their “development of chemical and biochemical analysis of DNA structure.”

2828

28-27

Replication in VitroReplication in Vitro• During replication, the sequence of nucleotides in one

strand is copied as a complementary strand to form the second strand of a double-stranded DNA

• Synthesis is catalyzed by DNA polymerase• DNA polymerase will catalyze synthesis in vitro using

single-stranded DNA as a template, provided that (1) the four deoxynucleotide triphosphate (dNTP) monomers and (2) a primer are present

• Primer:Primer: an oligonucleotide capable of forming a short section of double-stranded DNA (dsDNA) by base-pairing with its complement on a single-stranded DNA (ssDNA)

2828

28-28

Replication in VitroReplication in Vitro• Because a new DNA strand grows from its 5' to 3' end,

the primer must have a free 3'-OH group to which the first nucleotide of the growing chain is added

5' Single-stranded DNAT C A A C G T C TA G A

TCAGPrimer 5'

3'

3'HO

OH

direction of synthesiscatalyzed by DNA polymerase

dATP, dTTP, dCTP, dGTP

2828

28-29

Dideoxy Chain TerminationDideoxy Chain Termination• the key to the chain termination method is addition to

the synthesizing medium of a 2’,3’-dideoxynucleotide triphosphate (ddNTP)

• because a ddNTP has no 3’-OH, chain synthesis is terminated where a ddNTP becomes incorporated

-O-P-O-P-O-P-O-CH2

O-

O

O- O-

O

H

Base

H H

H HO

H

A 2',3'-dideoxynucleoside triphosphate (ddNTP)

O

2828

28-30

Dideoxy Chain TerminationDideoxy Chain TerminationIn this method, the following are mixed• single-stranded DNA of unknown sequence and

primer; then divided into four reaction mixtures

To each reaction mixture is then added• the four dNTP, one of which is labeled in the 5’ end

with phosphorus-32

• DNA polymerase• one of the four ddNTPs

15P32

16S32 + Beta particle+ Gamma rays

2828

28-31

Dideoxy Chain TerminationDideoxy Chain Terminationafter gel electrophoresis of each reaction mixture• a piece of film is placed over the gel• gamma rays released by P-32 darken the film and

create a pattern of the resolved oligonucleotide• the base sequence of the complement to the original

strand is read directly from bottom to top of the developed film

2828

28-32

Dideoxy Chain TerminationDideoxy Chain Termination

A G C T

Larger fragments

Smaller fragments

3'

5'

A

G

T

T

G

C

T

A

Thissequenceis the complementof the DNAtemplate

Polyacrylamide gel electrophoresis

If the complement of the DNA template is5'-A-T-C-G-T-T-G-A-3'Then the original DNA template must be5'-T-C-A-A-C-G-A-T-3'

2828

28-33

Prob 28.8Prob 28.8Draw structural formulas for the enethiol (the sulfur equivalent of an enol) forms of each antimetabolite used in the treatment of certain types of cancer.

6

6-Mercaptopurine6-Thioguanine

6HN

N

S

N

N

H

N

H

N

H2N

S

N

HN

2828

28-34

Prob 28.9Prob 28.9Draw two additional tautomers for cytosine and three additional tautomers for thymine.

Thymine (T)Cytosine (C)

N

N

H

O

NH2 O

O

H

HN

N

CH3

2828

28-35

Nucleic AcidsNucleic Acids

End Chapter 28End Chapter 28