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Chapter 28 Nucleosides, Nucleotides, and Nucleic Acids

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Title: Chapter 28 Nucleosides, Nucleotides, and Nucleic Acids


1
Chapter 28 Nucleosides, Nucleotides, and Nucleic
Acids
2
28.1 Pyrimidines and Purines
3
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.

4
Pyrimidines and Purines
  • Pyrimidine and purine are the names of the parent
    compounds of two types of nitrogen-containing
    heterocyclic aromatic compounds.

Pyrimidine
Purine
5
Pyrimidines and Purines
  • Amino-substituted derivatives of pyrimidine and
    purine have the structures expected from their
    names.

4-Aminopyrimidine
6-Aminopurine
6
Pyrimidines and Purines
  • But hydroxy-substituted pyrimidines and purines
    exist in keto, rather than enol, forms.

enol
keto
7
Pyrimidines and Purines
  • But hydroxy-substituted pyrimidines and purines
    exist in keto, rather than enol, forms.

enol
8
Important Pyrimidines
  • Pyrimidines that occur in DNA are cytosine and
    thymine. Cytosine and uracil are the pyrimidines
    in RNA.

NH2
HN
O
N H
Uracil
Thymine
Cytosine
9
Important Purines
  • Adenine and guanine are the principal purines of
    both DNA and RNA.

Adenine
Guanine
10
Caffeine and Theobromine
  • Caffeine (coffee) and theobromine (coffee and
    tea) are naturally occurring purines.

11
28.2 Nucleosides
12
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.

13
Table 28.2
  • Pyrimidine nucleosides

Cytidine
Cytidine occurs in RNA its 2-deoxy analog
occurs in DNA
14
Table 28.2
  • Pyrimidine nucleosides

Thymidine
Thymidine occurs in DNA
15
Table 28.2
  • Pyrimidine nucleosides

Uridine
Uridine occurs in RNA
16
Table 28.2
  • Purine nucleosides

Adenosine
Adenosine occurs in RNA its 2-deoxy analog
occurs in DNA
17
Table 28.2
  • Purine nucleosides

Guanosine
Guanosine occurs in RNA its 2-deoxy analog
occurs in DNA
18
28.3 Nucleotides
  • Nucleotides are phosphoric acid esters of
    nucleosides.

19
Adenosine 5'-Monophosphate (AMP)
  • Adenosine 5'-monophosphate (AMP) is also called
    5'-adenylic acid.

20
Adenosine Diphosphate (ADP)
21
Adenosine Triphosphate (ATP)
  • ATP is an important molecule in several
    biochemical processes including energy storage
    (Sections 28.4-28.5) phosphorylation

22
ATP and Phosphorylation
hexokinase
This is the first step in the metabolism of
glucose.
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.

Cyclic adenosine monophosphate (cAMP)
24
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.

Cyclic guanosine monophosphate (cGMP)
25
28.4 Bioenergetics
26
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

27
Standard Free Energy (DG)
  • 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.

28
Standard Free Energy (DG)
  • 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.

29
Standard Free Energy (DG)
  • 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'.

30
28.5 ATP and Bioenergetics
31
Hydrolysis of ATP
ATP H2O
ADP HPO42
  • ?G' for hydrolysis of ATP to ADP is 31 kJ/mol
  • Relative to ADP HPO42, ATP is a "high-energy"
    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.

32
Glutamic Acid to Glutamine
DG' 14 kJ
Reaction is endergonic
33
Glutamic Acid to Glutamine
OCCH2CH2CHCO
NH4
ATP
NH3
Reaction becomes exergonic when coupled to the
hydrolysis of ATP
34
Glutamic Acid to Glutamine
OCCH2CH2CHCO
ATP
NH3
Mechanism involves phosphorylation of glutamic
acid
35
Glutamic Acid to Glutamine
followed by reaction of phosphorylated glutamic
acid with ammonia
36
28.6 Phosphodiesters, Oligonucleotides, and
Polynucleotides
37
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.

38
Fig. 28.1 The trinucleotide ATG
  • phosphodiester linkages between 3' of one
    nucleotide and 5' of the next

39
28.7 Nucleic Acids
  • Nucleic acids are polynucleotides.

40
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.

41
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

42
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

43
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

44
28.8 Secondary Structure of DNA The Double Helix
45
Base Pairing
  • Watson and Crick proposed that A and T were
    present in equal amounts in DNA because of
    complementary hydrogen bonding.

46
Base Pairing
  • Watson and Crick proposed that A and T were
    present in equal amounts in DNA because of
    complementary hydrogen bonding.

47
Base Pairing
  • Likewise, the amounts of G and C in DNA were
    equal because of complementary hydrogen bonding.

48
Base Pairing
  • Likewise, the amounts of G and C in DNA were
    equal because of complementary hydrogen bonding.

49
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 (AT and GC)
  • 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.

50
Fig. 28.4
  • Two antiparallel strands of DNA are paired by
    hydrogen bonds between purine and pyrimidine
    bases.

51
Fig. 28.5
  • Helical structure of DNA. The purine and
    pyrimidine bases are on the inside, sugars and
    phosphates on the outside.

52
28.9 Tertiary Structure of DNA Supercoils
53
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.

54
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.

55
Histones
Each nucleosome contains one and three-quarters
turns of coil 146 base pairs. Linker contains
about 50 base pairs.
56
Histones
Nucleosome

57
28.10 Replication of DNA
58
Fig. 28.8 DNA Replication
  • The DNA to be copied is a double helix, shown
    here as flat for clarity.

The two strands begin to unwind. (next slide)
59
Fig. 28.8 DNA Replication
  • Each strand will become a template for
    construction of its complement.

60
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.

Polynucleotide chains grow in the 5'-3'
directioncontinuous in the leading strand,
discontinuous in the lagging strand.
61
Fig. 28.8 DNA Replication
  • Two duplex DNAs result, each of which is
    identical to the original DNA.

62
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.

63
Fig. 28.9 Chain elongation
64
Fig. 28.9 Chain elongation
65
28.11 Ribonucleic Acids
66
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 transla
    tion

67
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.

68
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.
69
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.

70
Table 28.3 (p 1175)
  • UUU Phe UCU Ser UAU Tyr UGU Cys U
  • UUC Phe UCC Ser UAC Tyr UGC Cys C
  • UUA Leu UCA Ser UAA Stop UGA Stop A
  • UUG Leu UCG Ser UAG Stop UCG Trp G
  • U
  • C
  • A
  • G
  • U
  • C
  • A
  • G
  • U
  • C
  • A
  • G

71
  • UUU Phe UCU Ser UAU Tyr UGU Cys U
  • UUC Phe UCC Ser UAC Tyr UGC Cys C
  • UUA Leu UCA Ser UAA Stop UGA Stop A
  • UUG Leu UCG Ser UAG Stop UCG Trp G
  • CUU Leu CCU Pro CAU His CGU Arg U
  • CUC Leu CCC Pro CAC His CGC Arg C
  • CUA Leu CCA Pro CAA Gln CGA Arg A
  • CUG Leu CCG Pro CAG Gln CCG Arg 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
  • GUU Val GCU Ala GAU Asp GGU Gly U
  • GUC Val GCC Ala GAC Asp GGC Gly C
  • GUA Val GCA Ala GAA Glu GGA Gly A
  • GUG Val GCG Ala GAG Glu GCG Gly G

72
  • U
  • C
  • UAA Stop UGA Stop A
  • UAG Stop G
  • U
  • 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
  • G

UAA, UGA, and UAG are "stop" codons that signal
the end of the polypeptide chain.
AUG is the "start" codon. Biosynthesis of
all proteins begins with methionine as the first
amino acid. This methionine is eventually
removed after protein synthesis is complete.
73
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.

74
Transfer RNA
  • ExamplePhenylalanine transfer RNA

One of the mRNA codons for phenylalanine is
75
Fig. 28.11 Phenylalanine tRNA
3'
3'
5'
5'
76
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 one-third
    protein.
  • It is believed that the ribosomal RNA acts as a
    catalysta ribozyme.

77
28.12 Protein Biosynthesis
78
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)

79
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.

80
Fig. 28.12 Translation
81
Fig. 28.12 Translation
  • Ester at 3' end of alanine tRNA is Met-Ala.
  • Process continues along mRNA until stop codon is
    reached.

82
28.13 AIDS
83
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)

84
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.

85
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.

86
AIDS Drugs
  • AZT and ddI are two drugs used against AIDS that
    delay onset of symptoms.

87
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.

88
28.14 DNA Sequencing
89
DNA Sequencing
  • Restriction enzymes cleave the polynucleotide to
    smaller fragments.
  • These smaller fragments (100-200 base pairs) are
    sequenced.
  • The two strands are separated.

90
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.

91
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.

92
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.

93
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.

94
Figure 27.29
Sequence of fragment
95
Figure 27.29
Sequence of fragment
Sequence of original DNA
96
28.15 The Human Genome Project
97
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.

98
28.16 DNA Profiling and the Polymerase Chain
Reaction
99
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.

100
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")

101
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 95C causes
the strands to separate. This is the
denaturation step.
102
Figure 28.14 (PCR)
(c) Cooling the sample to 60C 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 95C causes
the strands to separate. This is the
denaturation step.
103
Figure 28.14 (PCR)
(c) Cooling the sample to 60C 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 72C.
104
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 72C.
105
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.
106
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.
107
Figure 28.14 (PCR)
(f) Elongation of the primed fragments
completes the second PCR cycle.
108
Figure 28.14 (PCR)
The two contain only the target region and and
are the ones that increase disproportionately in
subsequent cycles.
109
Table 28.4
Cycle Total DNAs Contain only target 0
(start) 1 0 1 2 0 2 4 0 3 8 2 4 16 8 5 32 22 10 1,
024 1,004 20 1,048,566 1,048,526 30 1,073,741,824
1,073,741,764
110
End of Chapter 28
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