Download OC 28 Nucleic Acids

28
Organic
Chemistry
William H. Brown &
Christopher S. Foote
28-1
28
Nucleic
Acids
Chapter 28
28-2
28 Nucleic Acids
 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
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28 Purine/Pyrimidine Bases
O
3
N
2
N
5
HN
6
O
1
6
1
N
2
N
O
N
O
H
Thymine (T)
N
H
Cytosine (C)
O
N
N
N
N
N H2
7
5
CH3
HN
H
Uracil (U)
Pyrimidine
N H2
O
4
N
HN
8
N
4
3
Purine
N
H
9
N
N
H
Adenine (A)
H 2N
N
N
H
Guanine (G)
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28 Nucleosides
 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 b-glycosidic bond
uracil
O
HN
b-D-riboside
1
O
N
5'
HOCH2 O
H
H
4'
H
2'
3'
HO
OH
Uridine
a b-N-glycosidic
bond
1'
H
anomeric
carbon
28-5
28 Nucleotides
 Nucleotide:
a nucleoside in which a molecule of
phosphoric acid is esterified with an -OH of the
monosaccharide,
NH2
N
O
-
5'
O P O CH2
-
O
H
N
O
N
N
H
H
H
OH
OH
Adenosine 5'-monophosphate
(AMP)
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28 Nucleotides
Example 28.1 identify these nucleotides
O
N H2
N
(a)
O
O
O
O-P- O-P- O-CH2
O
O
O
H
H
H
HO
H
N
H 2N
(b) HOCH2
N
N
O
H
H
N
HN
H
H
O
H
O P O
O-
H
28-7
28 Acyclovir & AZT
O
N
HN
H2 N N
HOCH2 O
H
H
O
N
H H
Acyclovir
(drawn to show its
structural relationship
to 2-deoxyguanosine
CH3
HN
O
HOCH2
H
N
O
H
N3
H
H
H
Azidothymidine (AZT)
28-8
28 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 unit
 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
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28 DNA - 1° Structure
O
phosphorylated
5' end
O
-
CH3
HN
5'
O
O-P- O-C H2
O
H
H
N
O
O
O= P O
Ofree 3' end
H
O
H
N
NH
H
CH2
H
N
O
N H2
H
H
H 3'
OH
N
H
28-10
28 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
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28 T-A Base Pairing
A
major factor stabilizing the double helix
28-12
28 C-G Base Pairing
28-13
28 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
28 DNA - 3° Structure
 Tertiary
structure: the three-dimensional
arrangement of all atoms of nucleic acid,
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 (Fig 28.10)
 Histone: a protein, particularly rich in the basic
amino acids lysine and arginine, that is found
associated with DNA molecules
28-15
28 DNA - 3° Structure
 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
28-16
28 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 b-D-ribose rather than b2-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
28-17
28 RNA
 RNA
molecules are classified according to their
structure and function
Type
Molecular Weight
Range (g/mol)
mRNA
tRNA
rRNA
25,000 - 1,000,000
23,000 - 30,000
35,000 - 1,100,000
Number of Percentage
Nucleotides of Cell RNA
75 - 3,000
73 - 94
120 - 2904
2
16
82
 Ribosomal
RNA (rRNA): a ribonucleic acid found
in ribosomes, the site of protein synthesis
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28 RNA
 Transfer
RNA (tRNA) : a ribonucleic acid that
carries a specific amino acid to the site of
protein synthesis on ribosomes
O
t RNA -O- P-O- CH 2
O
amino acid, bound
as an ester to its
specific tRNA
-
H
H
O
O
Base
H
H
OH
C= O
C
N H3 +
H
R
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28 RNA
 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
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28 RNA
• the synthesis of mRNA from DNA is called
transcription
DNA template
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'
mRNA
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28 The Genetic Code
U
C
U
UUU Phe
UUC Phe
UUA Leu
UUG Leu
C
UCU Ser
UCC Ser
UCA Ser
UCG Ser
A
UAU
UAC
UAA
UAG
CUU Leu
CUC Leu
CUA Leu
CUG Leu
CCU Pro
CCC Pro
CCA Pro
CCG Pro
CAU His
CAC His
CAA Gln
CAG Gln
CGU Arg
CGC Arg
CGA Arg
CGG Arg
U
C
A
G
Ile
Ile
Ile
Met
ACU Thr
ACC Thr
ACA Thr
ACG Thr
AAU Asn
AAC Asn
AAA Lys
AAG Lys
AGU Ser
AGC Ser
AGA Arg
AGG Arg
U
C
A
G
GUU Val
GUC Val
GUA Val
GUG Val
GCU Ala
GCC Ala
GCA Ala
GCG Ala
GAU Asp
GAC Asp
GAA Glu
GAG Glu
GGU
GGC
GGA
GGG
U
C
A
G
AUU
A AUC
AUA
AUG*
G
G
Tyr UGU Cys
Tyr UGC Cys
Stop UGA Stop
Stop UGG Trp
*AUG also serves as the principal initiation codon.
Gly
Gly
Gly
Gly
U
C
A
G
28-22
28 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
• 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
28-23
28 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 from E. coli, for example, cleaves as shown
cleavage here
5'
G- A- A -T -T - C- - -3 '
EcoRI
5'
G + 5 ' -A - A- T -T -C- -- 3 '
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28 Sequencing DNA
• following are several more examples of
endonucleases and their specificities
Recognition
Enzyme Sequence
AluI
AG CT
BalI
TGG CCA
Recognition
Enzyme Sequence
HpaII C CGG
Mbol
GATC
FnuDII CG CG
Not I
GC GGCCGC
HeaIII GG CC
SacI
GAGCT C
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28 Sequencing DNA
 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: 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.”
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28 Replication 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: an oligonucleotide capable of forming a short
section of double-stranded DNA (dsDNA) by basepairing with its complement on a single-stranded DNA
(ssDNA)
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28 Replication 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'
T
C
A
A
Single-stranded DNA
C
G
A
T
C
G
dATP, dTTP, dCTP, dGTP
3' HO
direction of synthesis
catalyzed by DNA polymerase
T
G
A
C
Primer
A
T
OH 3'
5'
28-28
28 Dideoxy 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
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 triphosphate
(ddNTP)
28-29
28 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 reaction mixture is then added
• the four dNTP, one of which is labeled in the 5’ end
with phosphorus-32
32
15P
32
16S
+ Beta particle + Gamma rays
• DNA polymerase
• one of the four ddNTPs
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28 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
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28 Dideoxy Chain Termination
Polyacrylamide gel electrophoresis
A
G
Larger fragments
C
T
3'
A
G
T
T
G
This
sequence
is the
complement
of the DNA
template
C
T
Smaller fragments
If the complement of the DNA template is
Then the original DNA template must be
A
5'
5 ' -A -T - C-G- T- T -G- A -3 '
5 ' -T -C- A -A -C- G-A - T- 3 '
28-32
28 Prob 28.8
Draw structural formulas for the enethiol (the sulfur
equivalent of an enol) forms of each antimetabolite used
in the treatment of certain types of cancer.
S
S
HN
6
N
N
N
H
6-Mercaptopurine
HN
H2 N
6
N
N
N
H
6-Thioguanine
28-33
28 Prob 28.9
Draw two additional tautomers for cytosine and three
additional tautomers for thymine.
N H2
N
O
O
CH3
HN
N
H
Cytosine (C)
O
N
H
Thymine (T)
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28
Nucleic Acids
End Chapter 28
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