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Reginald H. Garrett
Charles M. Grisham
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Chapter 10
Nucleotides and Nucleic Acids
Reginald Garrett & Charles Grisham • University of Virginia
Chapter 10
“We have discovered the
secret of life.”
Francis Crick, to patrons of
Th E
The
Eagle,
l a pub
b iin
Cambridge, England (1953)
Francis Crick (right) and
James Watson (left)
point out features of their
model for the structure of
DNA.
Essential Questions
• What are the structures of the nucleotides?
• How
Ho are n
nucleotides
cleotides joined together to form
nucleic acids?
• How is information stored in nucleic acids?
• What are the biological functions of nucleotides
and nucleic acids?
Outline
• What are the structure and chemistry of nitrogenous
bases?
• What are nucleosides?
• What are the structure and chemistry of nucleotides?
• What are nucleic acids?
• What are the different classes of nucleic Acids?
• Are nucleic acids susceptible to hydrolysis?
Information Transfer in Cells
• Information encoded in a DNA molecule is
transcribed via synthesis of an RNA molecule
• The sequence of the RNA molecule is "read"
and is translated into the sequence of amino
acids in a protein
• See Figure 10.1
Information Transfer in Cells
Figure 10.1 The
fundamental process of
information transfer in
cells.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Know the basic structures
• Pyrimidines
• Cytosine (DNA, RNA)
• Uracil (RNA)
• Thymine (DNA)
• Purines
• Adenine
Ad i (DNA,
(DNA RNA)
• Guanine (DNA, RNA)
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.2 (a) The pyrimidine ring system; by convention,
atoms are numbered as indicated.
(b) The purine ring system; atoms numbered as shown.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.3 The common pyrimidine bases – cytosine,
uracil, and thymine – in the tautomeric forms
predominant at pH 7.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.4 The common purine bases – adenine and
guanine – in the tautomeric forms predominant at pH 7.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.5 Other naturally occurring
purine
i d
derivatives
i ti
– hypoxanthine,
h
thi
xanthine, and uric acid.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
• The aromaticity and electron-rich nature of
pyrimidines and purines enable them to undergo
keto-enol tautomerism
• The keto tautomers of uracil, thymine, and
guanine predominate at pH 7
• By contrast, the enol form of cytosine
predominates at pH 7
• Protonation
P t
ti states
t t off the
th nitrogens
it
determines
d t
i
whether they can serve as H-bond donors or
acceptors
• Aromaticity also accounts for strong absorption
of UV light
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
Figure 10.6 The keto-enol tautomerism of uracil.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
Figure 10.7 The tautomerization of the purine guanine.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
Figure 10.8 The UV absorption spectra of the common
ribonucleotides.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
Figure 10.8 The UV absorption spectra of the common
ribonucleotides.
10.2 What Are Nucleosides?
Structures to Know
• Nucleosides are compounds formed when a
b
base
iis lilinked
k d tto a sugar via
i a glycosidic
l
idi b
bond
d
• The sugars are pentoses
• D-ribose (in RNA)
• 2-deoxy-D-ribose (in DNA)
• The difference - 2'-OH vs 2'-H
• This difference affects secondary structure and
stability
10.2 What Are Nucleosides?
Figure 10.9 The linear and cyclic (furanose) forms of
ribose.
10.2 What Are Nucleosides?
Figure 10.9 The linear and cyclic (furanose) forms of
deoxyribose.
10.2 What Are Nucleosides?
• The base is linked to the sugar via a glycosidic bond
• The carbon of the glycosidic bond is anomeric
• Named by adding -idine
idine to the root name of a
pyrimidine or -osine to the root name of a purine
• Conformation can be syn or anti
• Sugars make nucleosides more water-soluble than
free bases
10.2 What Are Nucleosides?
Figure 10.10 The common ribonucleosides.
Adenosine: A Nucleoside with
Physiological Activity
• Adenosine functions as an
autacoid, or local hormone, and
neuromodulator.
• Circulating
C
in the bloodstream, it
influences blood vessel dilation,
smooth muscle contraction,
neurotransmitter release, and fat
metabolism.
• Adenosine is also a sleep
regulator Adenosine rises during
regulator.
wakefulness, promoting eventual
sleepiness.
• Caffeine promotes wakefulness
by blocking binding of adenosine
to its neuronal receptors.
10.3 What Is the Structure and Chemistry
of Nucleotides?
Nucleotides are nucleoside phosphates
• Know the nomenclature
• "Nucleotide phosphate" is redundant!
• Most nucleotides are ribonucleotides
• Nucleotides are polyprotic acids
10.3 What Is the Structure and Chemistry
of Nucleotides?
Figure 10.11 Structures of the four common ribonucleotides –
AMP, GMP, CMP, and UMP. Also shown: 3’-AMP.
10.3 What Is the Structure and Chemistry
of Nucleotides?
Figure 10.12 The cyclic nucleotide cAMP.
10.3 What Is the Structure and Chemistry
of Nucleotides?
Figure 10.12 The cyclic nucleotide cGMP
10.3 What Is the Structure and Chemistry
of Nucleotides?
Figure 10.13 Formation of ADP and ATP by the succesive
addition of phosphate groups via phosphoric anhydride
linkages. Note that the reaction is a dehydration synthesis.
10.3 What Is the Structure and Chemistry
of Nucleotides?
Figure 10.13 Formation of ADP and ATP by the succesive
addition of phosphate groups via phosphoric anhydride
linkages. Note that the reaction is a dehydration synthesis.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
• Nucleoside 5'-triphosphates are indispensable
agents in metabolism because their phosphoric
anhydride
y
bonds are a source of chemical energy
gy
• Bases serve as recognition units
• Cyclic nucleotides are signal molecules and
regulators of cellular metabolism and reproduction
• ATP is central to energy metabolism
• GTP drives p
protein synthesis
y
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl
group transfer, the major biochemical reactions of nucleotides.
Phosphoryl group transfer is shown here.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, and
nucleotidyl group transfer, the major biochemical
reactions of nucleotides. Pyrophosphoryl group
transfer is shown here.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, and
nucleotidyl group transfer, the major biochemical
reactions of nucleotides. Nucleotidyl group transfer is
shown here.
here
10.4 What Are Nucleic Acids?
• Nucleic acids are linear polymers of nucleotides
linked 3' to 5' by phosphodiester bridges
• Ribonucleic acid and deoxyribonucleic acid
• Know the shorthand notations
• Sequence is always read 5' to 3'
• In terms of genetic information, this
corresponds to "N to C" in proteins
10.4 What Are Nucleic Acids?
Figure
g
10.15 3',5',
Phosphodiester bridges link
nucleotides together to form
polynucleotide chains. The 5'ends of the chains are at the
top; the 3'-ends are at the
bottom. RNA is shown here.
10.4 What Are Nucleic Acids?
Figure 10
10.15
15 3’
3 ,5
5’phosphodiester bridges link
nucleotides together to form
polynucleotide chains. The
5’-ends of the chains are at
the top; the 3’-ends are at
the bottom. DNA is shown
here.
10.5 What Are the Different Classes of
Nucleic Acids?
• DNA - one type, one purpose
• RNA - 3 (or 4) types, 3 (or 4) purposes
• ribosomal
ib
l RNA - the
th basis
b i off structure
t t
and
d
function of ribosomes
• messenger RNA - carries the message for
protein synthesis
• transfer RNA - carries the amino acids for
protein synthesis
• Others:
• Small nuclear RNA
• Small non-coding RNAs
10.5 What Are the Different Classes of
Nucleic Acids?
Figure 10.16 The
antiparallel nature of the
DNA double helix. The two
chains have opposite
orientations.
The DNA Double Helix
The double helix is stabilized by hydrogen bonds
• "Base
Base pairs"
pairs arise from hydrogen bonds
A-T; G-C
•Erwin Chargaff had the pairing data, but didn't
understand its implications
•Rosalind Franklin's X-ray fiber diffraction data
was crucial
•Francis Crick showed that it was a helix
•James Watson figured out the H bonds
Chargaff’s Data Held the Clue to Base
Pairing
The Base Pairs Postulated by Watson
Figure 10.17 The Watson-Crick base pairs A:T and G:C.
A:T is shown here.
The Base Pairs Postulated by Watson
Figure 10.17 The Watson-Crick base pairs A:T and G:C.
G:C is shown here.
The Structure of DNA
An antiparallel double helix
•Diameter
Diameter of 2 nm
•Length of 1.6 million nm (E. coli)
•Compact and folded (E. coli cell is only 2000 nm
long)
•Eukaryotic DNA wrapped around histone
proteins to form nucleosomes
•Base pairs: A-T, G-C
The Structure of DNA
Figure 10.18 Replication of DNA
gives
g
es identical
de t ca p
progeny
oge y molecules
o ecu es
because base pairing is the
mechanism that determines the
nucleotide sequence of each newly
synthesized strand.
Digestion of the E. coli cell wall releases
the bacterial chromosome
Figure 10.19 The chromosome is shown surrounding the cell.
Do the Properties of DNA Invite Practical
Applications?
• The molecular recognition between DNA strands can
create a molecule with mechanical properties
different from single-stranded
g
DNA
• DNA double helices are relatively rigid rods
• DNA chains have been used to construct
nanomachines capable of simple movements such
as rotation and pincerlike motions
• More elaborate DNA-based devices can act as
motors walking along DNA tracks
• The construction of “DNA tweezers” is described on
the following slide
Do the Properties of DNA Invite Practical
Applications?
DNA tweezers – a simple
DNA nanomachine.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
Transcription product of DNA
prokaryotes,
y
a single
g mRNA contains the
• In p
information for synthesis of many proteins
• In eukaryotes, a single mRNA codes for just
one protein, but structure is composed of
introns and exons
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
Figure 10.20 Transcription and translation of mRNA
molecules in prokaryotic versus eukaryotic cells.
In prokaryotes, a single mRNA molecule may contain the
information for the synthesis of several polypeptide
chains within its nucleotide sequence.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
Figure 10.20 Transcription and translation of mRNA molecules
in prokaryotic versus eukaryotic cells.
Eukaryotic mRNAs encode only one polypeptide but are more
complex.
Eukaryotic mRNA
• DNA is transcribed to produce heterogeneous
nuclear RNA (hnRNA)
• mixed introns and exons with poly A
• intron = intervening sequence
• exon = coding sequence
• poly A tail - stability?
• Splicing produces final mRNA without introns
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
• Ribosomes are about 2/3 RNA, 1/3 protein
• rRNA serves as a scaffold for ribosomal proteins
• The different species of rRNA are referred to
according to their sedimentation coefficients
• rRNAs typically contain certain modified
nucleotides, including pseudouridine and
ribothymidylic acid
• The role of ribosomes in biosynthesis of proteins
is treated in detail in Chapter 30
• Briefly: the genetic information in the nucleotide
sequence of mRNA is translated into the amino
acid sequence of a polypeptide chain by
ribosomes
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
Figure 10.21 Ribosomal RNA
has a complex secondary
structure due to many
intrastrand H bonds. The gray
line here traces a
polynucleotide chain consisting
of more than 1000 nucleotides.
Aligned regions represent Hbonded complementary base
sequences.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
Figure 10.22 The organization and composition of ribosomes.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
Figure 1-23 Unusual bases in DNA.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
Figure 10.23 Unusual bases in DNA.
Transfer RNAs Carry Amino Acids to
Ribosomes for Use in Protein Synthesis
• Small polynucleotide chains - 73 to 94 residues
each
• Several bases usually methylated
• Each a.a. has at least one unique tRNA which
carries the a.a. to the ribosome
• 3'-terminal sequence is always CCA-3′-OH. The
a.a. is attached in ester linkage to this 3′-OH.
• Aminoacyl tRNA molecules are the substrates
off protein
t i synthesis
th i
Transfer RNAs Carry Amino Acids to
Ribosomes for Use in Protein Synthesis
Figure 10.24 Transfer RNA also
has a complex secondary
structure due to many intrastrand
hydrogen bonds. The black lines
represent base-paired
nucleotides in the sequence.
The RNA World and Early Evolution
• Thomas Cech and Sidney Altman showed that RNA
molecules are not only informational – they can also
be catalytic
y
• This gave evidence to the postulate by Francis Crick
and others that prebiotic evolution depended on selfreplicating, catalytic RNAs
• But what was the origin of the nucleotides?
• A likely source may have been conversion of
aminoimidazolecarbonitrile to adenine
• And glycolaldehyde could combine with other
molecules to form ribose
• Adenine and glycolaldehyde exist in outer space
The RNA World and Early Evolution
Aminoimidazolecarbonitrile is a pentamer of HCN and
may be a celestial precursor of adenine.
The RNA World and Early Evolution
Glycolaldehyde has been detected at the
center of the Milky Way and could be a
precursor of ribose and glucose.
The Chemical Differences Between DNA
and RNA Have Biological Significance
• Two fundamental chemical differences distinguish
DNA from RNA:
2 deoxyribose instead of ribose
• DNA contains 2-deoxyribose
• DNA contains thymine instead of uracil
The Chemical Differences Between DNA
and RNA Have Biological Significance
Why does DNA contain thymine?
• Cytosine spontaneously deaminates to form
uracil
• Repair enzymes recognize these "mutations"
and replace these Us with Cs
• But how would the repair enzymes distinguish
natural U from mutant U?
• Nature solves this dilemma byy using
g thymine
y
(5-methyl-U) in place of uracil
The Chemical Differences Between DNA
and RNA Have Biological Significance
Figure 10.25 Deamination of cytosine forms uracil.
The Chemical Differences Between DNA
and RNA Have Biological Significance
Figure 10.26 The 5-methyl
group on thymine labels it
as a special kind of uracil.
DNA & RNA Differences?
Why is DNA 2'-deoxy and RNA is not?
• Vicinal -OH groups (2' and 3') in RNA make it
more susceptible
tibl tto h
hydrolysis
d l i
• DNA, lacking 2'-OH is more stable
• This makes sense - the genetic material must
be more stable
• RNA is designed to be used and then broken
down
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
•
•
•
•
•
RNA is resistant to dilute acid
DNA is depurinated by dilute acid
DNA is not susceptible to base
RNA is hydrolyzed by dilute base
See Figure 10.27 for mechanism
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic
attach by OH- on the P atom leads to 5'-phosphoester
cleavage.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack by
OH- on the P atom leads to 5'-phosphoester cleavage. Random
hydrolysis of the cyclic phosphodiester intermediate gives a
mixture of 2'- and 3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Random hydrolysis of
the cyclic phosphodiester intermediate gives a mixture of 2'- and
3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10.28 Cleavage in polynucleotide chains. Cleavage
on the a side leaves the phosphate attached to the 5'position of the adjacent nucleotide. b-side hydrolysis yields
3'-phosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10.28 Cleavage in polynucleotide chains.
Cleavage
g on the a side leaves the p
phosphate
p
attached to
the 5'-position of the adjacent nucleotide.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
Figure 10
10.28
28 Cleavage in polynucleotide chains
chains.
b-side hydrolysis yields 3'-phosphate products, among
others.
Restriction Enzymes
• Bacteria have learned to "restrict" the
possibility of attack from foreign DNA by
means of "restriction
restriction enzymes"
enzymes
• Type II and III restriction enzymes cleave
DNA chains at selected sites
• Enzymes may recognize 4, 6 or more
bases in selecting sites for cleavage
• An enzyme that recognizes a 6-base
sequence is a "six-cutter"
Type II Restriction Enzymes
• No ATP requirement
• Recognition sites in dsDNA have a 2-fold axis of
symmetry
• Cleavage can leave staggered or "sticky" ends or
can produce "blunt” ends
Type II Restriction Enzymes
•
•
•
•
Names use 3-letter italicized code:
1st letter - genus; 2nd,3rd - species
F ll i lletter
Following
tt d
denotes
t strain
t i
EcoRI is the first restriction enzyme isolated from
the R strain of E. coli
Cleavage Sequences of Restriction
Endonucleases
Cleavage Sequences of Restriction
Endonucleases
Cleavage Sequences of Restriction
Endonucleases
Restriction Mapping of DNA
Figure 10.29
Restriction
mapping analysis.