Nucleic Acids
28-1
Nucleic Acids Components
 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.
28-2
Purine/Pyrimidine Bases
O
3
4
N
2
N
5
HN
6
O
1
6
1
N
2
N
O
N
O
H
Th ymin e (T)
N
H
C ytos i n e (C )
O
N
N
N
N
N H2
7
5
CH3
HN
H
Uraci l (U)
Pyri mi din e
N H2
O
N
HN
8
N
3
4
N
H
Pu rin e
9
N
N
H
Ade n in e (A)
H 2N
N
N
H
Gu an in e (G)
28-3
Nucleosides Definition
 Nucleoside:
A building block consisting of
• D-ribose or 2-deoxy-D-ribose
• heterocyclic aromatic amine base
• b-N-glycosidic bond .
u racil
O
HN
1
b-D -ribosid e
O
N
5'
HOCH2
H
4'
H
3'
O
H
2'
HO
OH
Uridine
a b-N-glycosid ic
bond
1'
H
an omeric
carb on
28-4
Nucleotides Definition
 Nucleotide:
Phosphoric acid ester of a
nucleoside, most commonly either the 3’ or the 5’
OH.
NH2
N
O
-
5'
O P O CH2
-
O
H
N
O
N
N
H
H
OH
OH
Aden os in e 5'-monophosp hate
(AMP)
H
28-5
Acyclovir & AZT
O
HN
H2 N N
HOCH2 O
H
H
O
N
CH3
HN
N
O
HOCH2
H H
A cyclovir
(drawn to s h ow i ts
s tru ctu ral re lation s h ip
to 2-de oxygu anos in e
H
N
O
H
N3
H
H
H
Az idoth ym idi n e (AZT)
Used to treat HIV
It is used to treat or prevent infections
caused by certain kinds of viruses.
Examples of these infections include
herpes and shingles
28-7
DNA - 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-Dribose is joined by a phosphodiester bond to the 5’-OH
of another 2-deoxy-D-ribose.
 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.
28-8
DNA - 1° Structure
• A structural formula for TG phosphorylated at the 5’
end.
O
ph osph oryl ate d
5' en d
HN
O
5'
O
O-P- O-C H2 O
O
H
3
H
diester
CH3
O
O= P O
Ofre e 3' e n d
Thymine, T
N
H
O Guanine,
H
N
NH
H
CH2
H
N
O
N
N H2
H
H
H 3'
OH
G
H
28-9
DNA - 2° 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: A type of 2° structure of DNA
molecules in which two antiparallel
polynucleotide strands are coiled in a righthanded manner about the same axis.
28-10
DNA - 2° Structure
• Hydrogen bonding occurs between bases
• A---T
• G---C
• Evidence: Base composition in mole-percent of DNA
for several organisms.
Purines
Organis m A
G
Human 30.4 19.9
Sheep
29.3 21.4
Yeast
31.7 18.3
E. coli
26.0 24.9
Pyrimidin es
C
T
19.9
21.0
17.4
25.2
30.1
28.3
32.6
23.9
A /T G/C
1.01 1.00
1.04 1.02
0.97 1.05
1.09 0.99
Purines/
Pyrimid ines
1.01
1.03
1.00
1.04
28-11
Base Pairing
• Base-pairing between adenine and thymine (A-T) and
guanine and cytosine (G-C).
28-12
Double Helix
• Ribbon model of double-stranded B-DNA.
28-13
Forms 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.
28-14
Double Helix
• An idealized model of B-DNA.
28-15
DNA - 3° Structure
 Tertiary
structure: The three-dimensional
arrangement of all atoms of a double-stranded
DNA, commonly referred as supercoiling.
 Circular DNA: A type of double-stranded DNA in
which the 5’ and 3’ ends of each stand are joined
by phosphodiester bonds.
 Histone: A protein, particularly rich in the basic
amino acids lysine and arginine, that is found
associated with DNA molecules.
28-16
DNA - 3° Structure
 Chromatin:
Consists of DNA molecules wound
around particles of histones (a simple protein
containing mainly basic amino acids;) in a beadlike
structure.
• Further coiling produces the dense chromatin found in
nuclei of plant and animal cells.
28-18
Ribonucleic Acids (RNA)
 RNA
• long, unbranched chains of nucleotides joined by
phosphodiester groups between the 3’-OH of one
pentose and the 5’-OH of the next;
• Consists of A, U ( Uracil), G, C.
• the pentose unit in RNA is b-D-ribose rather than b-2deoxy-D-ribose.
• the pyrimidine bases in RNA are uracil and cytosine
rather than thymine and cytosine.
• RNA is single stranded rather than double stranded.
(RNA)
A –Uracil
C –Cytosine
28-19
rRNA
 Different
types of RNA:
Type
Molecular Weight
Range (g/mol)
N umber of
N ucleotid es
mRN A
tRN A
rRN A
25,000 - 1,000,000
23,000 - 30,000
35,000 - 1,100,000
75 - 3,000
73 - 94
120 - 2904
Percen tage
of Cell RN A
2
16
82
 Ribosomal
RNA (rRNA): A ribonucleic acid found
in ribosomes, the site of protein synthesis.
28-20
tRNA
 Transfer
RNA (tRNA): A ribonucleic acid that
carries a specific amino acid to the site of protein
synthesis on ribosomes.
O
Base
t RNA -O- P-O- CH 2
O
H
H
O
H
H
ami n o aci d, bou n d
O
OH
as an e s te r to its
s pe ci fi c tRNA
C= O
C
N H3 +
H
R
28-21
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.
• mRNA 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.
28-22
mRNA from DNA, transcription
DNA (RNA)
A – T(U)
G – C(C)
28-23
The Genetic Code
second
U
UUU Ph e
UUC Ph e
UUA Le u
UUG Le u
C
UC U
UC C
UC A
UC G
C
C UU
C UC
C UA
C UG
Le u
Le u
Le u
Le u
A
AUU
AUC
AUA
AUG*
G
GUU
GUC
GUA
GUG
U
f
i
r
s
t
Ser
Se r
Ser
Ser
A
UAU
UAC
UAA
UAG
G
Tyr UGU
Tyr UGC
S top UGA
S top UGG
C ys
C ys
S top
Trp
U
C
A
G
CCU
CCC
CCA
CCG
Pro
Pro
Pro
Pro
C AU
C AC
C AA
C AG
Hi s
His
Gl n
Gl n
C GU
C GC
C GA
C GG
Arg
Arg
Arg
Arg
U
C
A
G
Il e
Ile
Il e
Me t
AC U
AC C
AC A
AC G
Th r
Th r
Th r
Th r
AAU
AAC
AAA
AAG
As n
As n
Lys
Lys
AGU
AGC
AGA
AGG
Ser
Ser
Arg
Arg
U
C
A
G
Val
Val
Val
Val
GC U
GC C
GC A
GC G
Al a
Al a
Al a
Al a
GAU
GAC
GAA
GAG
As p
As p
Gl u
Gl u
GGU
GGC
GGA
GGG
Gl y
Gl y
Gl y
Gl y
U
C
A
G
*AUG al s o se rves as th e prin cipal in i ti ati on codon .
t
h
i
r
d
28-24
The 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.
• Degenerate triplets differ only in the third letter of the
codon that varies. Gly, for example, is coded for by
GGA, GGG, GGC, and GGU. (GG? Codes for GLY)
• There is no ambiguity in the code; each triplet codes
for one and only one amino acid.
28-25
Sequencing DNA
 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 (eco R 1) from E. coli, for example, cleaves as
cle avage h e re
shown.
EcoRI
5'
G- A- A -T -T - C- - -3 '
Recognition
pattern
5'
G + 5 ' -A - A- T -T -C- -- 3 '
28-26
Sequencing DNA
• Following are several more examples of
endonucleases and their specificities.
Re cogn i ti on
En z ym e S e qu en ce
Alu I
AG C T
BalI
TGG C C A
Re cogn i ti on
En z ym e S e qu en ce
HpaII C C GG
Mbol
GA TC
FnuDII C G C G
N ot I
GC GGC C GC
HeaIII GG C C
SacI
GAG C T C
28-27
Sequencing DNA
• Maxam-Gilbert method: A method developed by Allan
Maxam and Walter Gilbert; depends on base-specific
chemical cleavage.
• 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.”
28-28
Replication in Vitro
• the sequence of nucleotides in one strand (ssDNA) is
copied as a complementary strand to form the second
strand of a double-stranded DNA (dsDNA).
• Synthesis is catalyzed by DNA polymerase.
• DNA polymerase requires
• the four deoxynucleotide triphosphate (dNTP) monomers
• a primer is present to start the process.
28-29
Dideoxy Chain Termination
• Chain termination method is accomplished by the
addition to the synthesizing medium of a 2’,3’dideoxynucleotide triphosphate (ddNTP).
• Because a ddNTP has no 3’-OH, chain synthesis is
terminated when a ddNTP becomes incorporated.
O
O
O
O- P-O- P-O- P-O-CH 2
O- O- OH
Base
O
H
H
H
H
H
A 2',3'-dideoxynucleoside tri phosphate
(ddNTP)
Without a OH here
chain cannot
extend.
28-30
Methodology of Dideoxy Chain Termination
In this method, the following are mixed:
• Single-stranded DNA of unknown sequence and
primer; then divided into four reaction mixtures.
 To
each of the four reaction mixture is then
added:
• The four dNTP, one of which is labeled in the 5’ end
with phosphorus-32 which is radioactive.
• DNA polymerase.
• one of the four ddNTPs.
28-31
Dideoxy Chain Termination
After 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.
28-32
Dideoxy Chain Termination
• The primer-DNA template is divided into four separate reaction
mixtures. To each is added the four dNTPs, DNA polymerase,
primer and one of the four ddNTPs in small amounts. Synthesis
will produce chains of varying lengths.
DNA
A–T
G–C
28-33
Dideoxy Chain Termination
The mixtures are separated by polyacrylamide gel
electrophoresis. From the four different ddNTP reaction mixtures.
Move
from 5’
to 3’
end.
28-34