Chapter 28: Nucleosides, Nucleotides, and Nucleic Acids.

Nucleic acids are the third class of biopolymers (polysaccharides

and proteins being the others)
Two major classes of nucleic acids
deoxyribonucleic acid (DNA): carrier of genetic information
ribonucleic acid (RNA): an intermediate in the expression
of genetic information and other diverse roles
The Central Dogma (F. Crick):
DNA
(genome)

mRNA
(transcriptome)

Protein
(proteome)

The monomeric units for nucleic acids are nucleotides

Nucleotides are made up of three structural subunits
1. Sugar: ribose in RNA, 2-deoxyribose in DNA
2. Heterocyclic base
3. Phosphate
1

Nucleoside, nucleotides and nucleic acids

phosphate
sugar
phosphate

phosphate
sugar

base
sugar

base

base

sugar

base

phosphate

nucleoside

nucleotides
sugar

base

nucleic acids

The chemical linkage between monomer units in nucleic acids

2
is a phosphodiester

28.1: Pyrimidines and Purines. The heterocyclic base; there

are five common bases for nucleic acids (Table 28.1, p. 1166).
Note that G, T and U exist in the keto form (and not the enol
form found in phenols)
7

NH2

8
9

N
H

N
H

N
N3

5
6

N1

N
H

NH2
N

pyrimidine

N
H

H3C
O

cytosine (C)
DNA/RNA

NH
N

NH2

guanine (G)
DNA/RNA
O

3
2

adenine (A)
DNA/RNA

purine

O
NH

N
H

thymine (T)
DNA

NH
N
H

uracil (U)
RNA

28.2: Nucleosides. N-Glycosides of a purine or pyrimidine

heterocyclic base and a carbohydrate. The C-N bond involves
the anomeric carbon of the carbohydrate. The carbohydrates for
nucleic acids are D-ribose and 2-deoxy-D-ribose
3

Nucleosides = carbohydrate + base (Table 28.2, p. 1168)

ribonucleosides or 2-deoxyribonucleosides
7

N
HO

5'

NH2
6

1
2

HO

1'

4'

HO

HO

RNA: X= OH, adenosine (A)

DNA: X= H, 2'-deoxyadenosine (dA)
4

HO

NH2

O
H3C

HO

O
NH

NH
HO

1'

4'
3'

RNA: X= OH, guanosine (G)

DNA: X= H, 2'-deoxyguanosine (dG)

NH2

5
5'

2'

3'

HO

NH

2'

RNA: X= OH, cytidine (C)

DNA: X= H, 2'-deoxycytidine (dC)

HO

DNA: thymidine (T)

HO

OH

RNA: R= H, uridine (U)

To differentiate the atoms of the carbohydrate from the base, the

position number of the carbohydrate is followed by a (prime).
The stereochemistry of the glycosidic bond found in nucleic acids
4
is .

28.3: Nucleotides. Phosphoric acid esters of nucleosides.

Nucleotides = nucleoside + phosphate
HO
HO

O
HO P O
O

O
X

HO

HO

O
O
O
HO P O P O P O
O
O
O

O
X

O
HO

ribonucleotide 5'-diphosphate (X=OH, NDP)

deoxyribonucleotide 5'-diphosphate (X=H, dNDP)

ribonucleotide 5'-monophosphate (X=OH, NMP)

deoxyribonucleotide 5'-monophosphate (X=H, dNMP)

ribonucleoside (X=OH)
deoxyribonucleoside (X=H)
O
O
HO P O P O
O
O

O
X

ribonucleotide 5'-triphosphate (NTP)

deoxyribonucleotide 5'-triphosphate (X=H, dNTP)

P
O

OH

ribonucleotide
3',5'-cyclic phosphosphate (cNMP)

Kinase: enzymes that catalyze the phosphoryl transfer reaction

from ATP to an acceptor substrate. M2+ dependent
HO
HO

O
OH
Glucose

OH

NH2

O
O
O
O P O P O P O
O
O
O

OH

N
N

HO

OH
ATP

HO
HO

OPO32O
OH

OH

Glucose-6-phosphate

NH2

O
O
O P O P O
O
O

N
N

HO

OH

ADP

NH2

O
O
O
O P O P O P O
O
O
O

N
HO

-P2O7

OH
O

O
O
O
O P O P O P O
O
O
O

N
HO

OH

NH
NH2

GTP

O
P
O

N
N

HO

OH

AMP
O

N
N

NH2

O
O P O
O

H2O

OH
cAMP
N

guanylate
cyclase
-P2O7

P
O

ATP

NH2

adenylyl
cyclase

OH
cGMP

NH
NH2

O
O P O
O

H2 O

N
N

HO

OH

NH
NH2

GMP

1971 Nobel Prize in Medicine or Physiology:

Earl Sutherland

28.4: Bioenergetics. (Please read)

28.5: ATP and Bioenergetics. (Please read)
NH2

O
O
O
O P O P O P O
O
O
O

N
N

HO

OH
ATP

H2O

NH2

O
O
O P O P O
O
O

N
HO

HPO4 2-

+ ~31 KJ/mol
(7.4 Kcal/mol)

OH
ADP

28.6: Phosphodiesters, Oligonucleotides, and

Polynucleotides. The chemical linkage between
nucleotide units in nucleic acids is a phosphodiester,
which connects the 5-hydroxyl group of one
nucleotide to the 3-hydroxyl group of
the next nucleotide.
By convention, nucleic acid sequences are written
from left to right, from the 5-end to the 3-end.
Nucleic acids are negatively charged

3'

O P
O
O
O

5'

Base

3'

O P
O
O

5'

Base

3'

5'

RNA, X= OH
DNA, X=H

28.7: Nucleic Acids.

1944: Avery, MacLeod & McCarty - Strong evidence that DNA
is genetic material
1950: Chargaff - careful analysis of DNA from a wide variety of
organisms. Content of A,T, C & G varied widely according
to the organism, however: A=T and C=G (Chargaff Rule)
1953: Watson & Crick - structure of DNA (1962 Nobel Prize with
7
M. Wilkens, 1962)

28.8: Secondary Structure of DNA: The Double Helix.

Initial like-with-like, parallel helix:
Does not fit with with Chargaffs Rule: A = T
G=C
H
H N
N

N
N

dR

H N
H

dR

N
H

N H

N
dR

N H

purine - purine

dR
N

H
N

dR

N
N
dR

N
O

dR
N

N
dR

pyrimidine - pyrimidine

Wrongtautomers!!

Watson,J.D.TheDoubleHelix,1968

Two polynucleotide strands, running in opposite directions

(anti-parallel) and coiled around each other in a double helix.
The strands are held together by complementary hydrogenbonding between specific pairs of bases.
AntiparallelCGPair
Hydrogen Bond
Donor

Hydrogen Bond
N3
Acceptor
Hydrogen Bond
Acceptor

N4

O2

N
N

5'

RO

O
H

5'
N

Bond
O6 Hydrogen
Acceptor

N
O

N1 Hydrogen Bond

RO

Donor

N
H

N2

Hydrogen Bond
Donor

N6

Hydrogen Bond
Donor

OR

3'

OR

3'

AntiparallelTAPair
Hydrogen Bond
Acceptor

O4

Hydrogen Bond
N3
Donor

5'
RO

OR
3'

N H
O

H N
N

H
N

5'

N
N

RO
O
OR
3'

N1 Hydrogen Bond
Acceptor

DNA double helix

major
groove
12
one
helical
turn
34

minor
groove
6

backbone: deoxyribose and phosphodiester linkage

bases

10

28.9: Tertiary Structure of DNA: Supercoils. Each cell contains

about two meters of DNA. DNA is packaged by coiling around
a core of proteins known as histones. The DNA-histone
assembly is called a nucleosome. Histones are rich is lysine
and arginine residues.

11
Pdb code 1kx5

It has not escaped our attention that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material. Watson & Crick

28.10: Replication of DNA.

The Central Dogma (F. Crick):
DNA

DNA transcription mRNA translation Protein

(genome)
(transcriptome)
(proteome)

replication

Expression and transfer of genetic information:

Replication: process by which DNA is copied with very high
fidelity.
Transcription: process by which the DNA genetic code is read
and transferred to messenger RNA (mRNA). This is an
intermediate step in protein expression
Translation: The process by which the genetic code is converted
to a protein, the end product of gene expression.
The DNA sequence codes for the mRNA sequence, which
codes for the protein sequence

12

DNA is replicated by the coordinated efforts of a number of

proteins and enzymes.
For replication, DNA must be unknotted, uncoiled and the
double helix unwound.
Topoisomerase: Enzyme that unknots and uncoils DNA
Helicase: Protein that unwinds the DNA double helix.
DNA polymerase: Enzyme that replicates DNA using each strand
as a template for the newly synthesized strand.
DNA ligase: enzyme that catalyzes the formation of the
phosphodiester bond between pieces of DNA.
DNA replication is semi-conservative: Each new strand of DNA
contains one parental (old, template) strand and one
daughter (newly synthesized) strand
13

Unwinding of DNA by helicases expose the DNA bases

(replication fork) so that replication can take place. Helicase
hydrolyzes ATP in order to break the hydrogen bonds between
DNA strands

DNA replication

14

DNA Polymerase: the new strand is replicated from the 5 3

(start from the 3-end of the template)
DNA polymerases are Mg2+ ion dependent
The deoxynucleotide 5-triphosphate (dNTP) is the reagent for
nucleotide incorporation
dNTP

OH
5'

O
O O
O P O P O P OO- O- O-

O
T

A
O

O
O

-O

template
strand
(old)

Mg2+

OH

G
O

C
5'

(new)

O
3'

3-hydroxyl group of the growing DNA strand acts as a nucleophile

and attacks the -phosphorus atom of the dNTP.
15

Replication of the leading strand occurs continuously in the

5 3 direction of the new strand.
Replication of the lagging strand occurs discontinuously. Short
DNA fragments are initially synthesized and then ligated together.
DNA ligase catalyzes the formation of the phosphodiester bond
between pieces of DNA.

animations of DNA processing: http://www.wehi.edu.au/education/wehi-tv/dna/

16

DNA replication occurs with very high fidelity:

Most DNA polymerases have high intrinsic fidelity
Many DNA polymerases have proof-reading
(exonuclease) activity
Mismatch repair proteins seek out and repair base-pair
mismatches due to unfaithful replication
28.11 Ribonucleic Acid
RNA contains ribose rather than 2-deoxyribose and uracil rather
than thymine. RNA usually exist as a single strand.
There are three major kinds of RNA
messenger RNA (mRNA):
ribosomal RNA (rRNA)
transfer RNA (tRNA)
DNA is found in the cell nucleus and mitochondria; RNA is more
17
disperse in the cell.

Transcription: only one of the DNA strands is copied (coding

or antisense strand). An RNA polymerase replicates the DNA
sequence into a complementary sequence of mRNA (template
or sense strand). mRNAs are transported from the nucleus to
the cytoplasm, where they acts as the template for protein
biosynthesis (translation). A three base segment of mRNA
(codon) codes for an amino acid.The reading frame of the
codons is defined by the start and stop codons.

18

5'-cap

5'-UTR

start

Coding sequence

stop

3'-UTR

The mRNA is positioned in the ribosome through complementary

pairing of the 5-untranslated region of mRNA with a rRNA.
Transfer RNA (tRNA): t-RNAs carries an amino acid on the
3-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome
catalyzes amide bond formation.
Ribosome: large assembly of proteins and rRNAs that catalyzes
protein and peptide biosynthesis using specific, complementary,
anti-parallel pairing interactions between mRNA and the
anti-codon loop of specific tRNAs.

19

Although single-stranded, there are complementary sequences

within tRNA that give it a defined conformation.
The three base codon sequence of mRNA are complementary
to the anti-codon loops of the appropriate tRNA. The basepairing between the mRNA and the tRNA positions the tRNAs
for amino acid transfer to the growing peptide chain.

variable loop
TC loop

D loop

aminoacyl t-RNA
20

28.12: Protein Biosynthesis. Ribosomal protein synthesis

SCH3
OHC

SCH3

H
N
OHC
O

H
N
O

OH

H2N
O

A site

P site

U A C
A U G U A U G C U C

U A C

U U

3'

5'

A U A

A U G U A U G C U C

U U

3'

5'
SCH33

CH3S

CH33S
OHC
OH

N
H

OJHC
OHC
HN

OH
OHC

OH

HN
N
H

HN
OH O

OH
O
HHN
2N

HN
O

OH
O

O
O

OH
CH33
CH
O
O

A U A
U A C
A
A U A C
C G
G A

U A C A
U A

5'

E site

A U G U A U G C U C U U

3'

5'
5'

A
U U
A U
U G
G U
U A
A U
U G
G C
C U
U C U

3'

21

28.13: AIDS. (please read)

28.14: DNA Sequencing.
Maxam-Gilbert: relys on reagents that react with a specific DNA
base that can subsequent give rise to a sequence
specific cleavage of DNA
Sanger: Enzymatic replication of the DNA fragment to be
sequenced with a DNA polymerase, Mg+2, and
dideoxynucleotides triphosphate (ddNTP) that truncates
DNA replication
Restriction endonucleases: Bacterial enzymes that cleave
DNA at specific sequences
5d(GAATTC)3
3d(CTTAAG)5

5d(GGATCC)3
3d(CCTAGG)5

5'

3'

3'

5'

EcoRI

restriction
enzyme

2-O PO
3

OH
5'

BAMHI

3'

2-O PO
3

3'
5'
OH

22

Sanger Sequencing:
key reagent: dideoxynucleotides triphosphates (ddNTP)
NH2
N
O
-O

P O P O P O
O-

O-

N
N

OddATP

3'

NH
O
-

P O P O P O
O-

O-

OddTTP

N
O

NH2

O
-O

P O P O P O
O-

O-

O-

NH
N

Mg2+, dNTP's
+ one ddATP

P O P O P O
O-

O-

ddCTP

Anti-Viral Nucleosides

When a ddNTP
is incorporated
elongation of
the primer is
terminated
The ddNTP is
specifically
incorporated
opposite its
complementary
nucleotide base

O
NH
HO

N
HO

NH

N3

AZT

ddI
NH2

HO

H2N

OH

O
S

d4C

(-)-3TC

5'

Template
unknown sequence

O-

ddGTP

primer

DNA polymerase

-O

5'-32P

3'

NH2

23

SangerSequencing
Larger
fragments

Smaller
fragments

ddA

ddG

ddC

ddT
5'

C
A
T
T
G
C
A
T
T
A
G
T
G
T
C

G
T
A
A
C
G
T
A
A
T
C
A
C
A
G

5'

3'

32

3'

32P-5'

primer

3'

template
24

28.15: The Human Genome Project. (please read)

28.16: DNA Profiling and Polymerase Chain Reaction (PCR).
method for amplifying DNA using a DNA polymerase, dNTPs and
cycling the temperature.
Heat stable DNA Polymerases (from archaea):
Taq: thermophilic bacteria (hot springs)- no proof reading
Pfu: geothermic vent bacteria- proof reading
Mg 2+
two Primer DNA strands (synthetic, large excess)
one sense primer and one antisense primer
one Template DNA strand (double strand)
dNTPs
1x2=2x2=4x2=8x2=16x2=32x2=64x2=128x2=256x2=512x2
=1,024x2=2,048x2=4,096x2=8,192x2=16,384x2=32,768x2=65,536x2
=131,072x2=262,144x2=524,288x2=1,048,576
In principle, over one million copies per original, can be obtained after just twenty cycles
KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the
polymerase chain reaction (PCR) method.

25

Polymerase Chain Reaction

5'

3'

3'

5'

3'

3'

5'

95 C
denaturation

5'

3'

55 - 68 C

extension
5'

3'

5'

3'

3'

5'

5'
3'

72 C
Taq, Mg 2+, dNTPs

3'

3'

anneal
(+) and (-) primers

5'

95 C

3'

denaturation

5'

2nd cycle

5'

3'

3'

5'

5'

3'

3'

5'

2 copies of DNA
repeat temperature cycles

amplification of DNA
For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html
http://users.ugent.be/~avierstr/principles/pcrani.html

26