Download Nucleosides, Nucleotides,Nucleic Acids

Chapter 28
Nucleosides, Nucleotides,
and Nucleic Acids
Dr. Wolf's CHM 424
28- 1
28.1
Pyrimidines and Purines
Dr. Wolf's CHM 424
28- 2
Pyrimidines and Purines
In order to understand the structure and
properties of DNA and RNA, we need to look at
their structural components.
We begin with certain heterocyclic aromatic
compounds called pyrimidines and purines.
Dr. Wolf's CHM 424
28- 3
Pyrimidines and Purines
Pyrimidine and purine are the names of the
parent compounds of two types of nitrogencontaining heterocyclic aromatic compounds.
6
N1
5
4
N
2
3
Pyrimidine
Dr. Wolf's CHM 424
7
N
5
N
H
4
6
N1
8
9
N
2
3
Purine
28- 4
Pyrimidines and Purines
Amino-substituted derivatives of pyrimidine and
purine have the structures expected from their
names.
H
H
NH2
N
N
N
H
H2N
N
H
N
N
H
H
4-Aminopyrimidine
Dr. Wolf's CHM 424
6-Aminopurine
28- 5
Pyrimidines and Purines
But hydroxy-substituted pyrimidines and purines
exist in keto, rather than enol, forms.
H
H
HO
H
H
N
N
H
O
N
N
H
H
enol
Dr. Wolf's CHM 424
keto
28- 6
Pyrimidines and Purines
But hydroxy-substituted pyrimidines and purines
exist in keto, rather than enol, forms.
OH
N
N
H
N
H
N
H
N
H
H
H
enol
Dr. Wolf's CHM 424
N
N
H
N
O
keto
28- 7
Important Pyrimidines
Pyrimidines that occur in DNA are cytosine and
thymine. Cytosine and uracil are the
pyrimidines in RNA.
O
O
NH2
CH3
HN
HN
HN
O
N
H
Uracil
Dr. Wolf's CHM 424
O
N
H
Thymine
O
N
H
Cytosine
28- 8
Important Purines
Adenine and guanine are the principal purines
of both DNA and RNA.
NH2
O
N
N
N
N
H
Adenine
Dr. Wolf's CHM 424
N
HN
H2N
N
N
H
Guanine
28- 9
Caffeine and Theobromine
Caffeine (coffee) and theobromine (coffee and
tea) are naturally occurring purines.
O
H3C
O
N
N
N
N
CH3
Caffeine
Dr. Wolf's CHM 424
O
CH3
N
HN
O
CH3
N
N
CH3
Theobromine
28- 10
28.2
Nucleosides
Dr. Wolf's CHM 424
28- 11
Nucleosides
The classical structural definition is that a
nucleoside is a pyrimidine or purine N-glycoside
of D-ribofuranose or 2-deoxy-D-ribofuranose.
Informal use has extended this definition to
apply to purine or pyrimidine N-glycosides of
almost any carbohydrate.
The purine or pyrimidine part of a nucleoside is
referred to as a purine or pyrimidine base.
Dr. Wolf's CHM 424
28- 12
Table 28.2
Pyrimidine nucleosides
NH2
N
N
Cytidine
HOCH2
HO
O
O
OH
Cytidine occurs in RNA;
its 2-deoxy analog occurs in DNA
Dr. Wolf's CHM 424
28- 13
Table 28.2
Pyrimidine nucleosides
O
H3C
Thymidine
HOCH2
NH
N
O
O
HO
Thymidine occurs in DNA
Dr. Wolf's CHM 424
28- 14
Table 28.2
Pyrimidine nucleosides
O
NH
N
Uridine
HOCH2
HO
O
O
OH
Uridine occurs in RNA
Dr. Wolf's CHM 424
28- 15
Table 28.2
Purine nucleosides
NH2
N
Adenosine
HOCH2 O
HO
N
N
N
OH
Adenosine occurs in RNA;
its 2-deoxy analog occurs in DNA
Dr. Wolf's CHM 424
28- 16
Table 28.2
Purine nucleosides
O
N
Guanosine
HOCH2 O
HO
Dr. Wolf's CHM 424
N
NH
N
NH2
OH
Guanosine occurs in RNA;
its 2-deoxy analog occurs in DNA
28- 17
28.3
Nucleotides
Nucleotides are phosphoric acid esters of
nucleosides.
Dr. Wolf's CHM 424
28- 18
Adenosine 5'-Monophosphate (AMP)
Adenosine 5'-monophosphate (AMP) is also
called 5'-adenylic acid.
NH2
N
N
O
5'
HO
P OCH2 O
HO
N
1'
4'
3'
HO
Dr. Wolf's CHM 424
N
2'
OH
28- 19
Adenosine Diphosphate (ADP)
NH2
N
O
HO
P O
HO
O
P OCH2 O
N
N
HO
HO
Dr. Wolf's CHM 424
N
OH
28- 20
Adenosine Triphosphate (ATP)
ATP is an important molecule in several
biochemical processes including:
energy storage (Sections 28.4-28.5)
phosphorylation
NH2
N
O
HO
O
P
O P O
HO
HO
O
P OCH2 O
N
N
HO
HO
Dr. Wolf's CHM 424
N
OH
28- 21
ATP and Phosphorylation
HOCH2
ATP +
HO
HO
O
HO
This is the first step in the
metabolism of glucose.
OH
hexokinase
O
ADP +
Dr. Wolf's CHM 424
(HO)2POCH2
O
HO
HO
HO
OH
28- 22
cAMP and cGMP
Cyclic AMP and cyclic GMP are
"second messengers" in many
biological processes. Hormones
(the "first messengers")
stimulate the formation of cAMP
and cGMP.
NH2
N
CH2 O
O
O
HO
N
N
N
P
O
OH
Cyclic adenosine monophosphate (cAMP)
Dr. Wolf's CHM 424
28- 23
cAMP and cGMP
Cyclic AMP and cyclic GMP are
"second messengers" in many
biological processes. Hormones
(the "first messengers")
stimulate the formation of cAMP
and cGMP.
O
O
HO
O
N
CH2 O
N
NH
N
NH2
P
O
OH
Cyclic guanosine monophosphate (cGMP)
Dr. Wolf's CHM 424
28- 24
28.4
Bioenergetics
Dr. Wolf's CHM 424
28- 25
Bioenergetics
Bioenergetics is the thermodynamics of
biological processes.
Emphasis is on free energy changes (DG)
when DG is negative, reaction is
spontaneous in the direction written
when DG is 0, reaction is at equilibrium
when DG is positive, reaction is not
spontaneous in direction written
Dr. Wolf's CHM 424
28- 26
Standard Free Energy (DG°)
mA(aq)
nB(aq)
Sign and magnitude of DG depends on what the
reactants and products are and their
concentrations.
In order to focus on reactants and products,
define a standard state.
The standard concentration is 1 M (for a
reaction in homogeneous solution).
DG in the standard state is called the standard
free-energy change and given the symbol DG°.
Dr. Wolf's CHM 424
28- 27
Standard Free Energy (DG°)
mA(aq)
nB(aq)
Exergonic: An exergonic reaction is one for
which the sign of DG° is negative.
Endergonic: An exergonic reaction is one for
which the sign of DG° is positive.
Dr. Wolf's CHM 424
28- 28
Standard Free Energy (DG°)
mA(aq)
nB(aq)
It is useful to define a special standard state
for biological reactions.
This special standard state is one for which
the pH = 7.
The free-energy change for a process under
these conditions is symbolized as DG°'.
Dr. Wolf's CHM 424
28- 29
28.5
ATP and Bioenergetics
Dr. Wolf's CHM 424
28- 30
Hydrolysis of ATP
ATP + H2O
ADP + HPO42–
DG°' for hydrolysis of ATP to ADP is –31 kJ/mol
Relative to ADP + HPO42–, ATP is a "highenergy" compound.
When coupled to some other process, the
conversion of ATP to ADP can provide the free
energy to transform an endergonic process to
an exergonic one.
Dr. Wolf's CHM 424
28- 31
Glutamic Acid to Glutamine
O
–OCCH
O
–
CH
CHCO
2
2
+ NH4+
+NH
3
DG°' = +14 kJ
O
Reaction is endergonic
O
H2NCCH2CH2CHCO–
+ H2O
+NH
3
Dr. Wolf's CHM 424
28- 32
Glutamic Acid to Glutamine
O
O
–OCCH CH CHCO–
2
2
+ NH4+
+ ATP
+NH
3
Reaction becomes exergonic
when coupled to the hydrolysis
of ATP
DG°' = –17 kJ
O
O
H2NCCH2CH2CHCO–
+ HPO42–
+ ADP
+NH
3
Dr. Wolf's CHM 424
28- 33
Glutamic Acid to Glutamine
O
O
–OCCH CH CHCO–
2
2
+ ATP
+NH
3
Mechanism involves
phosphorylation of glutamic
acid
O
–O
P
–O
Dr. Wolf's CHM 424
O
O
OCCH2CH2CHCO–
+ ADP
+NH
3
28- 34
Glutamic Acid to Glutamine
O
O
H2NCCH2CH2CHCO–
+ HPO42–
+NH
3
O
–O
P
–O
Dr. Wolf's CHM 424
O
followed by reaction of
phosphorylated glutamic acid
with ammonia
O
OCCH2CH2CHCO–
+ NH3
+NH
3
28- 35
28.6
Phosphodiesters,
Oligonucleotides, and
Polynucleotides
Dr. Wolf's CHM 424
28- 36
Phosphodiesters
A phosphodiester linkage between two
nucleotides is analogous to a peptide bond
between two amino acids.
Two nucleotides joined by a phosphodiester
linkage gives a dinucleotide.
Three nucleotides joined by two
phosphodiester linkages gives a trinucleotide,
etc. (See next slide)
A polynucleotide of about 50 or fewer
nucleotides is called an oligonucleotide.
Dr. Wolf's CHM 424
28- 37
NH2
free 5' end
N
Fig. 28.1
The
trinucleotide
ATG
N
5'
HOCH2
O
phosphodiester
linkages
between 3' of
one nucleotide
and 5' of the
next
A
N
O
H3C
O
O P OCH2
O
T HO
NH
N
O
O
N
G
free 3' end
Dr. Wolf's CHM 424
N
O
O P OCH2
O
HO
N
NH
N
NH2
3'
HO
28- 38
28.7
Nucleic Acids
Nucleic acids are polynucleotides.
Dr. Wolf's CHM 424
28- 39
Nucleic Acids
Nucleic acids first isolated in 1869 (Johann
Miescher)
Oswald Avery discovered (1945) that a
substance which caused a change in the
genetically transmitted characteristics of a
bacterium was DNA.
Scientists revised their opinion of the function of
DNA and began to suspect it was the major
functional component of genes.
Dr. Wolf's CHM 424
28- 40
Composition of DNA
Erwin Chargaff (Columbia Univ.) studied DNAs
from various sources and analyzed the
distribution of purines and pyrimidines in them.
The distribution of the bases adenine (A),
guanine (G), thymine (T), and cytosine (C)
varied among species.
But the total purines (A and G) and the total
pyrimidines (T and C) were always equal.
Moreover: %A = %T, and %G = %C
Dr. Wolf's CHM 424
28- 41
Composition of Human DNA
For example:
Purine
Pyrimidine
Adenine (A) 30.3%
Thymine (T) 30.3%
Guanine (G) 19.5%
Cytosine (C) 19.9%
Total purines: 49.8% Total pyrimidines: 50.1%
Dr. Wolf's CHM 424
28- 42
Structure of DNA
James D. Watson and Francis H. C. Crick
proposed a structure for DNA in 1953.
Watson and Crick's structure was based on:
•Chargaff's observations
•X-ray crystallographic data of Maurice
Wilkins and Rosalind Franklin
•Model building
Dr. Wolf's CHM 424
28- 43
28.8
Secondary Structure of DNA:
The Double Helix
Dr. Wolf's CHM 424
28- 44
Base Pairing
Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.
H
N
N
N
N
H
O
H
CH3
N
2-deoxyribose N
N
O
A
Dr. Wolf's CHM 424
T
2-deoxyribose
28- 45
Base Pairing
Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.
Dr. Wolf's CHM 424
28- 46
Base Pairing
Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.
H
O
N
H N
N
2-deoxyribose
N H
N
N
N H
H
G
Dr. Wolf's CHM 424
N
O
2-deoxyribose
C
28- 47
Base Pairing
Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.
Dr. Wolf's CHM 424
28- 48
The DNA Duplex
Watson and Crick proposed a double-stranded
structure for DNA in which a purine or
pyrimidine base in one chain is hydrogen
bonded to its complement in the other.
•Gives proper Chargaff ratios (A=T and G=C)
•Because each pair contains one purine and
one pyrimidine, the A---T and G---C distances
between strands are approximately equal.
•Complementarity between strands suggests a
mechanism for copying genetic information.
Dr. Wolf's CHM 424
28- 49
O
3'
Fig. 28.4
O
C
5'
O
5'
G
O
Two antiparallel
strands of DNA
are paired by
hydrogen bonds
between purine
and pyrimidine
bases.
O
O P O
3' O
3'
Ğ
O P O
O
T
5'
A
O
O P O
O
T
5'
A
O
O P O
O
O
O P O
3' O
3'
Ğ
Ğ
O
5'
O
O
O P O
3' O
3'
Ğ
Ğ
O
5'
O
O
Ğ
O
G
5'
C
5'
O
O
3'
O
Dr. Wolf's CHM 424
28- 50
Fig. 28.5
Helical structure of
DNA. The purine
and pyrimidine
bases are on the
inside, sugars and
phosphates on the
outside.
Dr. Wolf's CHM 424
28- 51
28.9
Tertiary Structure of DNA:
Supercoils
Dr. Wolf's CHM 424
28- 52
DNA is coiled
A strand of DNA is too long (about 3 cm in
length) to fit inside a cell unless it is coiled.
Random coiling would reduce accessibility to
critical regions.
Efficient coiling of DNA is accomplished with the
aid of proteins called histones.
Dr. Wolf's CHM 424
28- 53
Histones
Histones are proteins rich in basic amino acids
such as lysine and arginine.
Histones are positively charged at biological pH.
DNA is negatively charged.
DNA winds around histone proteins to form
nucleosomes.
Dr. Wolf's CHM 424
28- 54
Histones
Each nucleosome contains one and three-quarters
turns of coil = 146 base pairs.
Linker contains about 50 base pairs.
Dr. Wolf's CHM 424
28- 55
Histones
Nucleosome =
Dr. Wolf's CHM 424
Histone proteins + Supercoiled DNA
28- 56
28.10
Replication of DNA
Dr. Wolf's CHM 424
28- 57
Fig. 28.8 DNA Replication
The DNA to be copied is a double helix, shown here as
flat for clarity.
5'
3'
T
A
A
T
G
C
G
C
C
G
A
T
T
A
C
G
C
G
T
A
G
C
A
T
A
T
T
A
C
G
5'
3'
The two strands begin to unwind. (next slide)
Dr. Wolf's CHM 424
28- 58
Fig. 28.8 DNA Replication
Each strand will become a template for construction of
its complement.
3'
5'
T
A
3'
A
T
G
C
G
C
C
G
A
T
T
A
C
G
C
T
G
A
G
C
A
T
A
T
T
A
C
G
5'
Dr. Wolf's CHM 424
28- 59
Fig. 28.8 DNA Replication
Two new strands form as nucleotides that are
complementary to those of the original strands are
joined by phosphodiester linkages.
3'
5'
T
A
G
G
C
A
T
C
T
C
A'
G
C'
A
T'
A
T'
T
A'
C
G'
5'
leading strand
3'
A
3'
T
C
C
G
T
A
5'
G
G
Polynucleotide chains grow in
the 5'-3' direction—continuous in
the leading strand, discontinuous
theCHM
lagging
strand.
Dr.in
Wolf's
424
A
C
A'
T
lagging strand
A'
T
T'
C'
A G
5'
3'
28- 60
Fig. 28.8 DNA Replication
Two duplex DNAs result, each of which is identical to
the original DNA.
5'
3'
T
A'
A
T'
G
C'
G
C'
C A
G' T'
T
A'
C
G'
C
G'
T
A'
G
C'
A
T'
A
T'
T
A'
C
G'
5'
3'
+
5'
3'
T'
A
3'
Dr. Wolf's CHM 424
A'
T
G' G' C'
C C G
A'
T
T'
A
C'
G
C'
G
T'
A
G'
C
A'
T
A'
T
T'
A
C'
G
5'
28- 61
Elongation of the growing DNA chain
The free 3'-OH group of the growing DNA chain
reacts with the 5'-triphosphate of the appropriate
nucleotide.
Dr. Wolf's CHM 424
28- 62
Fig. 28.9 Chain elongation
OH
Adenine,
Guanine,
Cytosine, or
Thymine
Adenine,
Guanine,
Cytosine, or
Thymine
Dr. Wolf's CHM 424
O
O
O
O
CH2 O P
O P
O P
O–
••
•• OH
O
O
CH2OPO
O–
O–
O–
Polynucleotide
chain
O–
28- 63
Fig. 28.9 Chain elongation
OH
Adenine,
Guanine,
Cytosine, or
Thymine
O
CH2
Adenine,
Guanine,
Cytosine, or
Thymine
Dr. Wolf's CHM 424
O
O
O
O
–O P
O P
O–
O–
O–
P
O–
•• O ••
O
O
CH2OPO
Polynucleotide
chain
O–
28- 64
28.11
Ribonucleic Acids
Dr. Wolf's CHM 424
28- 65
DNA and Protein Biosynthesis
According to Crick, the "central dogma" of
molecular biology is:
"DNA makes RNA makes protein."
Three kinds of RNA are involved.
messenger RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
There are two main stages.
transcription
translation
Dr. Wolf's CHM 424
28- 66
Transcription
In transcription, a strand of DNA acts as a
template upon which a complementary RNA is
biosynthesized.
This complementary RNA is messenger RNA
(mRNA).
Mechanism of transcription resembles
mechanism of DNA replication.
Transcription begins at the 5' end of DNA and is
catalyzed by the enzyme RNA polymerase.
Dr. Wolf's CHM 424
28- 67
Fig. 28.10 Transcription
Only a section of about 10 base pairs in the DNA
is unwound at a time. Nucleotides complementary
to the DNA are added to form mRNA.
Dr. Wolf's CHM 424
28- 68
The Genetic Code
The nucleotide sequence of mRNA codes for
the different amino acids found in proteins.
There are three nucleotides per codon.
There are 64 possible combinations of A, U, G,
and C.
The genetic code is redundant. Some proteins
are coded for by more than one codon.
Dr. Wolf's CHM 424
28- 69
U
U
UUU
UUC
UUA
UUG
Phe
Phe
Leu
Leu
C
UCU
UCC
UCA
UCG
Ser
Ser
Ser
Ser
A
UAU
UAC
UAA
UAG
Tyr
Tyr
Stop
Stop
C
First letter
A
Second letter
Third letter
G
UGU
UGC
UGA
UCG
Cys
Cys
Stop
Trp
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
G
Table 28.3 (p 1175)
Dr. Wolf's CHM 424
28- 70
U
U
C
A
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
C
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
A
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
G
UGU
UGC
UGA
UCG
CGU
CGC
CGA
CCG
AGU
AGC
AGA
ACG
GGU
GGC
GGA
GCG
Cys
Cys
Stop
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
G
Dr. Wolf's CHM 424
28- 71
U
U
C
A
C
A
G
U
C
UAA, UGA, and UAG
UAA Stop
UGA Stop A
are "stop" codons that
UAG Stop
G
signal the end of the
U
polypeptide chain.
C
A
G
AUU Ile
ACU Thr
AAU Asn
AGU Ser U
AUC Ile
ACC Thr
AAC Asn
AGC Ser C
AUA Ile
ACA Thr
AAA Lys
AGA Arg A
AUG Met
ACG Thr
AAG Lys
ACG Arg G
U
C
A
AUG is the "start" codon. Biosynthesis of all
G
proteins begins with methionine as the first amino
G acid. This methionine is eventually removed after
protein synthesis is complete.
Dr. Wolf's CHM 424
28- 72
Transfer tRNA
There are 20 different tRNAs, one for each
amino acid.
Each tRNA is single stranded with a CCA triplet
at its 3' end.
A particular amino acid is attached to the tRNA
by an ester linkage involving the carboxyl group
of the amino acid and the 3' oxygen of the
tRNA.
Dr. Wolf's CHM 424
28- 73
Transfer RNA
Example—Phenylalanine transfer RNA
One of the mRNA codons for phenylalanine is:
5'
UUC
3'
The complementary sequence in tRNA is called
the anticodon.
3'
Dr. Wolf's CHM 424
AAG
5'
28- 74
Fig. 28.11 Phenylalanine tRNA
O
OCCHCH2C6H5
3'
NH3
+
3'
5'
5'
Anticodon
Dr. Wolf's CHM 424
28- 75
Ribosomal RNA
Most of the RNA in a cell is ribosomal RNA
Ribosomes are the site of protein synthesis.
They are where translation of the mRNA
sequence to an amino acid sequence occurs.
Ribosomes are about two-thirds RNA and onethird protein.
It is believed that the ribosomal RNA acts as a
catalyst—a ribozyme.
Dr. Wolf's CHM 424
28- 76
28.12
Protein Biosynthesis
Dr. Wolf's CHM 424
28- 77
Protein Biosynthesis
During translation the protein is synthesized
beginning at its N-terminus.
mRNA is read in its 5'-3' direction
begins at the start codon AUG
ends at stop codon (UAA, UAG, or UGA)
Dr. Wolf's CHM 424
28- 78
Fig. 28.12 Translation
Methionine at N-terminus
is present as its N-formyl
derivative.
Reaction that occurs is
nucleophilic acyl
substitution. Ester is
converted to amide.
Dr. Wolf's CHM 424
28- 79
Fig. 28.12 Translation
Dr. Wolf's CHM 424
28- 80
Fig. 28.12 Translation
Ester at 3' end of
alanine tRNA is Met-Ala.
Process continues
along mRNA until stop
codon is reached.
Dr. Wolf's CHM 424
28- 81
28.13
AIDS
Dr. Wolf's CHM 424
28- 82
AIDS
Acquired immune deficiency syndrome
More than 22 million people have died from
AIDS since disease discovered in 1980s
Now fourth leading cause of death worldwide
and leading cause of death in Africa (World
Health Organization)
Dr. Wolf's CHM 424
28- 83
HIV
Virus responsible for AIDS in people is HIV
(human immunodeficiency virus)
Several strains of HIV designated HIV-1, HIV-2,
etc.
HIV is a retrovirus. Genetic material is RNA, not
DNA.
Dr. Wolf's CHM 424
28- 84
HIV
HIV inserts its own RNA and an enzyme
(reverse transcriptase) in T4 lymphocyte cell of
host.
Reverse transcriptase catalyzes the formation of
DNA complementary to the HIV RNA.
HIV reproduces and eventually infects other T4
lympocytes.
Ability of T4 cells to reproduce decreases,
interfering with bodies ability to fight infection.
Dr. Wolf's CHM 424
28- 85
AIDS Drugs
AZT and ddI are two drugs used against AIDS
that delay onset of symptoms.
O
O
H3 C
HOCH2
O
O
N3
AZT
Dr. Wolf's CHM 424
N
NH
N
O
NH
HOCH2
N N
O
O
H
H
H
H
ddI
28- 86
AIDS Drugs
Protease inhibitors are used in conjunction with
other AIDS drugs.
Several HIV proteins are present in the same
polypeptide chain and must be separated from
each other in order to act.
Protease inhibitors prevent formation of HIV
proteins by preventing hydrolysis of polypeptide
that incorporates them.
Dr. Wolf's CHM 424
28- 87
28.14
DNA Sequencing
Dr. Wolf's CHM 424
28- 88
DNA Sequencing
Restriction enzymes cleave the polynucleotide
to smaller fragments.
These smaller fragments (100-200 base pairs)
are sequenced.
The two strands are separated.
Dr. Wolf's CHM 424
28- 89
DNA Sequencing
Single stranded DNA divided in four portions.
Each tube contains adenosine, thymidine,
guanosine, and cytidine plus the triphosphates
of their 2'-deoxy analogs.
OH
OH
HO
P
O
O
P
O
OH
O
O
O
HO
Dr. Wolf's CHM 424
base
POCH2
H
28- 90
DNA Sequencing
The first tube also contains the 2,'3'-dideoxy analog of
adenosine triphosphate (ddATP); the second tube the
2,'3'-dideoxy analog of thymidine triphosphate (ddTTP),
the third contains ddGTP, and the fourth ddCTP.
OH
OH
HO
P
O
O
P
O
OH
O
O
O
H
Dr. Wolf's CHM 424
base
POCH2
H
28- 91
DNA Sequencing
Each tube also contains a "primer," a short
section of the complementary DNA strand,
labeled with radioactive phosphorus (32P).
DNA synthesis takes place, producing a
complementary strand of the DNA strand used
as a template.
DNA synthesis stops when a dideoxynucleotide
is incorporated into the growing chain.
Dr. Wolf's CHM 424
28- 92
DNA Sequencing
The contents of each tube are separated by
electrophoresis and analyzed by
autoradiography.
There are four lanes on the electrophoresis gel.
Each DNA fragment will be one nucleotide
longer than the previous one.
Dr. Wolf's CHM 424
28- 93
Figure 27.29
ddA
ddT
ddG
ddC
Sequence of
fragment
T
TG
TGA
TGAC
TGACA
TGACAT
TGACATA
TGACATAC
TGACATACG
TGACATACGT
Dr. Wolf's CHM 424
28- 94
Figure 27.29
ddA
ddT
Dr. Wolf's CHM 424
ddG
ddC
Sequence of
fragment
Sequence of
original DNA
T
A
TG
AC
TGA
ACT
TGAC
ACTG
TGACA
ACTGT
TGACAT
ACTGTA
TGACATA
ACTGTAT
TGACATAC
ACTGTATG
TGACATACG
ACTGTATGC
TGACATACGT
ACTGTATGCA
28- 95
28.15
The Human Genome Project
Dr. Wolf's CHM 424
28- 96
Human Genome Project
In 1988 National Research Council (NRC)
recommended that the U.S. undertake the
mapping and sequencing of the human
genome.
International Human Genome Sequencing
Consortium (led by U.S. NIH) and Celera
Genomics undertook project. Orginally
competitors, they agreed to coordinate efforts
and published draft sequences in 2001.
Dr. Wolf's CHM 424
28- 97
28.16
DNA Profiling
and the
Polymerase Chain Reaction
Dr. Wolf's CHM 424
28- 98
DNA Profiling
DNA sequencing involves determining the
nucleotide sequence in DNA.
The nucleotide sequence in regions of DNA that
code for proteins varies little from one individual
to another, because the proteins are the same.
Most of the nucleotides in DNA are in
"noncoding" regions and vary significantly
among individuals.
Enzymatic cleavage of DNA give a mixture of
polynucleotides that can be separated by
electrophoresis to give a "profile" characteristic
of a single individual.
Dr. Wolf's CHM 424
28- 99
PCR
When a sample of DNA is too small to be
sequenced or profiled, the polymerase chain
reaction (PCR) is used to make copies
("amplify") portions of it.
PCR amplifies DNA by repetitive cycles of the
following steps.
1. Denaturation
2. Annealing ("priming")
3. Synthesis ("extension" or "elongation")
Dr. Wolf's CHM 424
28- 100
Figure 28.14 (PCR)
(a) Consider double-stranded DNA containing
a polynucleotide sequence (the target region)
that you wish to amplify.
Target region
(b) Heating the DNA to about 95°C causes the
strands to separate. This is the denaturation
step.
Dr. Wolf's CHM 424
28- 101
Figure 28.14 (PCR)
(c) Cooling the sample to ~60°C causes one
primer oligonucleotide to bind to one strand and
the other primer to the other strand. This is the
annealing step.
(b) Heating the DNA to about 95°C causes the
strands to separate. This is the denaturation
step.
Dr. Wolf's CHM 424
28- 102
Figure 28.14 (PCR)
(c) Cooling the sample to ~60°C causes one
primer oligonucleotide to bind to one strand and
the other primer to the other strand. This is the
annealing step.
(d) In the presence of four DNA nucleotides and
the enzyme DNA polymerase, the primer is
extended in its 3' direction. This is the synthesis
step and is carried out at 72°C.
Dr. Wolf's CHM 424
28- 103
Figure 28.14 (PCR)
This completes one cycle of PCR.
(d) In the presence of four DNA nucleotides and
the enzyme DNA polymerase, the primer is
extended in its 3' direction. This is the synthesis
step and is carried out at 72°C.
Dr. Wolf's CHM 424
28- 104
Figure 28.14 (PCR)
This completes one cycle of PCR.
(e) The next cycle begins with the denaturation
of the two DNA molecules shown. Both are
then primed as before.
Dr. Wolf's CHM 424
28- 105
Figure 28.14 (PCR)
(f) Elongation of the primed fragments completes
the second PCR cycle.
(e) The next cycle begins with the denaturation
of the two DNA molecules shown. Both are
then primed as before.
Dr. Wolf's CHM 424
28- 106
Figure 28.14 (PCR)
(f) Elongation of the primed fragments completes
the second PCR cycle.
(g) Among the 8 DNAs formed in the second
cycle are two having the structure shown.
Dr. Wolf's CHM 424
28- 107
Figure 28.14 (PCR)
The two contain only the target region and
and are the ones that increase disproportionately
in subsequent cycles.
(g) Among the 8 DNAs formed in the second
cycle are two having the structure shown.
Dr. Wolf's CHM 424
28- 108
Table 28.4
Cycle
Total DNAs
0 (start)
1
1
2
2
4
3
8
4
16
5
32
10
1,024
20
1,048,566
30
1,073,741,824
Dr. Wolf's CHM 424
Contain only target
0
0
0
2
8
22
1,004
1,048,526
1,073,741,764
28- 109
End of Chapter 28
Dr. Wolf's CHM 424
28- 110