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Chapter 26
Nucleosides, Nucleotides,
and Nucleic Acids
26.1
Pyrimidines and Purines
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.
Pyrimidines and Purines
Pyrimidine and purine are the names of the
parent compounds of two types of nitrogencontaining heterocyclic aromatic compounds.
6
7
N1
5
4
N
2
3
Pyrimidine
N
5
N
H
4
6
N1
8
9
N
3
Purine
2
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
6-Aminopurine
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
enol
keto
H
Pyrimidines and Purines
But hydroxy-substituted pyrimidines and purines
exist in keto, rather than enol, forms.
OH
N
N
N
H
N
O
N
H
H
N
H
H
enol
N
keto
N
H
H
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
O
N
H
Thymine
O
N
H
Cytosine
Important Purines
Adenine and guanine are the principal purines
of both DNA and RNA.
NH2
O
N
N
N
N
H
Adenine
N
HN
H2N
N
N
H
Guanine
Caffeine and Theobromine
Caffeine (coffee) and theobromine (coffee and
tea) are naturally occurring purines.
O
H3C
O
N
N
N
O
CH3
N
CH3
Caffeine
N
HN
O
CH3
N
N
CH3
Theobromine
26.2
Nucleosides
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.
Table 26.2
Pyrimidine nucleosides
NH2
N
N
Cytidine
HOCH2
HO
O
O
OH
Cytidine occurs in RNA;
its 2-deoxy analog occurs in DNA.
Table 26.2
Pyrimidine nucleosides
O
H3C
Thymidine
HOCH2
NH
N
O
O
HO
Thymidine occurs in DNA.
Table 26.2
Pyrimidine nucleosides
O
NH
N
Uridine
HOCH2
HO
O
O
OH
Uridine occurs in RNA.
Table 26.2
Purine nucleosides
NH2
N
Adenosine
HOCH2 O
HO
N
N
N
OH
Adenosine occurs in RNA;
its 2-deoxy analog occurs in DNA.
Table 26.2
Purine nucleosides
O
N
Guanosine
HOCH2 O
HO
N
NH
N
NH2
OH
Guanosine occurs in RNA;
its 2-deoxy analog occurs in DNA.
26.3
Nucleotides
Nucleotides are phosphoric acid esters of
nucleosides.
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
2'
OH
N
Adenosine Diphosphate (ADP)
NH2
N
O
HO
P O
HO
N
O
P OCH2 O
N
HO
HO
OH
N
Adenosine Triphosphate (ATP)
ATP is an important molecule in several
biochemical processes including:
energy storage (Sections 26.4-26.5)
phosphorylation
NH2
N
O
HO
O
P
O P O
HO
HO
N
O
P OCH2 O
N
HO
HO
OH
N
ATP and Phosphorylation
HOCH2
ATP +
HO
HO
O
HO
This is the first step in the
metabolism of glucose.
OH
hexokinase
O
ADP +
(HO)2POCH2
O
HO
HO
HO
OH
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)
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)
26.4
Bioenergetics
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.
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°.
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.
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°'.
26.5
ATP and Bioenergetics
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.
Glutamic Acid to Glutamine
O
–OCCH
O
–
CH
CHCO
2
2
+ NH4+
+NH
3
DG°' = +14 kJ
O
Reaction is endergonic.
O
H2NCCH2CH2CHCO–
+NH
3
+ H2O
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–
+NH
3
+ HPO42–
+ ADP
Glutamic Acid to Glutamine
O
O
–OCCH CH CHCO–
2
2
+ ATP
+NH
3
Mechanism involves
phosphorylation of glutamic
acid.
O
–O
P
–O
O
O
OCCH2CH2CHCO–
+NH
3
+ ADP
Glutamic Acid to Glutamine
O
O
H2NCCH2CH2CHCO–
+ HPO42–
+NH
3
O
–O
P
–O
O
Followed by reaction of
phosphorylated glutamic acid
with ammonia.
O
OCCH2CH2CHCO–
+NH
3
+ NH3
26.6
Phosphodiesters,
Oligonucleotides, and
Polynucleotides
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.
Fig. 26.1
The
trinucleotide
ATG
free 5' end
phosphodiester
linkages
between 3' of
one nucleotide
and 5' of the
next
A
T
G
free 3' end
26.7
Nucleic Acids
Nucleic acids are polynucleotides.
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.
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
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%
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
26.8
Secondary Structure of DNA:
The Double Helix
Base Pairing
Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.
2-deoxyribose
A
T
2-deoxyribose
Base Pairing
Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.
Base Pairing
Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.
2-deoxyribose
2-deoxyribose
G
C
Base Pairing
Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.
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.
O
3'
Fig. 26.4
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
G
5'
O
3'
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
C
5'
O
Ğ
Fig. 26.5
Helical structure of
DNA. The purine
and pyrimidine
bases are on the
inside, sugars and
phosphates on the
outside.
26.9
Tertiary Structure of DNA:
Supercoils
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.
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.
Histones
Each nucleosome contains one and three-quarters
turns of coil = 146 base pairs.
Linker contains about 50 base pairs.
Histones
Nucleosome =
Histone proteins + Supercoiled DNA
26.10
Replication of DNA
Fig. 26.8 DNA Replication
Fig. 26.8 DNA Replication
Fig. 26.8 DNA Replication
Fig. 26.8 DNA Replication
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.
Fig. 26.9: Chain Elongation
OH
Adenine,
Guanine,
Cytosine, or
Thymine
Adenine,
Guanine,
Cytosine, or
Thymine
O
O
O
O
CH2 O P
O P
O P
O–
••
•• OH
O
O
CH2OPO
O–
O–
Polynucleotide
chain
O–
O–
Fig. 26.9: Chain Elongation
OH
Adenine,
Guanine,
Cytosine, or
Thymine
O
CH2
Adenine,
Guanine,
Cytosine, or
Thymine
O
O
O
O
–O P
O P
O–
O–
O–
P
O–
•• O ••
O
O
CH2OPO
O–
Polynucleotide
chain
26.11
Ribonucleic Acids
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
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.
Fig. 26.10: 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.
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.
U
U
UUU
UUC
UUA
UUG
Phe
Phe
Leu
Leu
C
UCU
UCC
UCA
UCG
Ser
Ser
Ser
Ser
A
UAU
UAC
UAA
UAG
C
First letter
A
Second letter
Third letter
G
Table 26.4
Tyr
Tyr
Stop
Stop
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
U
UUU
UUC
U
UUA
UUG
CUU
CUC
C
CUA
CUG
AUU
AUC
A
AUA
AUG
GUU
GUC
G 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
U
C
A
G
U
UAA, UGA, and UAG
C
U are "stop" codons that
UAA Stop
UGA Stop A
signal the end of the
UAG Stop
G
polypeptide chain.
U
C
C
A
G
AUU Ile
ACU Thr
AAU Asn
AGU Ser U
AUC Ile
ACC Thr
AAC Asn
AGC Ser C
A
AUA Ile
ACA Thr
AAA Lys
AGA Arg A
AUG Met
ACG Thr
AAG Lys
ACG Arg G
U
AUG is the "start" codon. Biosynthesis of all
proteins begins with methionine as the first amino C
G acid. This methionine is eventually removed after A
G
protein synthesis is complete.
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.
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'
AAG
5'
Fig. 26.11: Phenylalanine tRNA
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.
26.12
Protein Biosynthesis
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)
Fig. 26.12: Translation
Methionine at N-terminus
is present as its N-formyl
derivative.
Reaction that occurs is
nucleophilic acyl
substitution. Ester is
converted to amide.
Fig. 26.12: Translation
Fig. 26.12: Translation
Ester at 3' end of
alanine tRNA is Met-Ala.
Process continues
along mRNA until stop
codon is reached.
26.13
AIDS
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).
HIV
Virus responsible for AIDS in people is Human
Immunodeficiency Virus (HIV).
Several strains of HIV designated HIV-1, HIV-2,
etc.
HIV is a retrovirus. Genetic material is RNA, not
DNA.
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.
AIDS Drugs
AZT and ddI are two drugs used against AIDS
that delay onset of symptoms.
O
O
H3 C
N
NH
HOCH2
O
N3
AZT
N
O
HOCH2
N N
O
H
H
H
H
ddI
NH
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.
26.14
DNA Sequencing
DNA Sequencing
Restriction enzymes cleave the polynucleotide
to smaller fragments.
These smaller fragments (100-200 base pairs)
are sequenced.
The two strands are separated.
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
OH
HO
P
O
O
P
O
O
base
POCH2
O
O
HO
H
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
base
POCH2
O
O
H
H
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.
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.
Figure 26.13
Figure 26.13
26.15
The Human Genome Project
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. Originally
competitors, they agreed to coordinate efforts
and published draft sequences in 2001.
26.16
DNA Profiling
and the
Polymerase Chain Reaction
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.
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")
Figure 26.14: (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.
Figure 26.14: (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.
Figure 26.14: (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.
Figure 26.14: (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.
Figure 26.14: (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.
Figure 26.14: (PCR)
(f) Elongation of the primed fragments completes
the second PCR cycle.
Figure 26.14: (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.
Figure 26.14: (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.
Table 26.5
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
Contain only target
0
0
0
2
8
22
1,004
1,048,526
1,073,741,764
26.17
Recombinant DNA Technology