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Chapter 10
Reginald H. Garrett
Charles M. Grisham
Chapter 10
Nucleotides and Nucleic Acids
“We have discovered the
secret of life.”
Francis Crick, to patrons of
The Eagle, a pub in
Cambridge, England (1953)
Nobel Prize 1962
Francis Crick (right) and
James Watson (left)
point out features of their
model for the structure of
DNA.
Information Transfer in Cells
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
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
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.
• Pyrimidines
• Cytosine (DNA, RNA)
• Uracil (RNA)
• Thymine (DNA)
• Purines
• Adenine (DNA, RNA)
• Guanine (DNA, RNA)
(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
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.5 Other naturally occurring
purine derivatives – hypoxanthine,
xanthine, and uric acid.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.4 The common purine bases – adenine and
guanine.
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 (互變異構性)
• The keto tautomers (互變異構物) of uracil,
thymine, and guanine predominate at pH 7
• By contrast, the enol form of cytosine
predominates at pH 7
• Protonation states of the nitrogens determines
whether they can serve as H-bond donors or
acceptors
• Aromaticity also accounts for strong absorption
of UV light quantitative & qualitative analysis
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
Figure 10.6 The keto-enol tautomerism of uracil.
Figure 10.8 The UV absorption spectra of the common
ribonucleotides.
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.
10.2 What Are Nucleosides?
Adenosine: A Nucleoside with
Physiological Activity
• Adenosine functions as an autacoid
(內分泌物) , or local hormone, and
neuromodulator (神經調節物質).
• Circulating 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 extended
wakefulness, promoting eventual
sleepiness.
• Caffeine promotes wakefulness by
blocking binding of adenosine to its
neuronal receptors.
10.2 What Are Nucleosides?
Structures to Know
• Nucleosides are compounds formed when a
base is linked to a sugar via a glycosidic bond
• 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?
• The base is linked to the sugar via a glycosidic
bond
• Named by adding -idine to the root name of a
pyrimidine or -osine to the root name of a purine
• Sugars make nucleosides more water-soluble than
free bases
Figure 10.9 The linear and cyclic (furanose) forms of
deoxyribose.
10.2 What Are Nucleosides?
10.3 What Is the Structure and Chemistry
of Nucleotides?
Nucleotides are nucleoside phosphates
•Know the nomenclature
• "Nucleotide phosphate" is redundant! wrong!
Uracil
Adenine
• Most monomeric (單體) nucleotides in the cells
are ribonucleotides having 5’-phosphate groups
Cytosine
(Fig. 10.11).
•Nucleoside diphosphates and triphosphates are
nucleotides with two or three phosphate groups
Guanine
Figure 10.10 The common ribonucleosides.
10.3 What Is the Structure and Chemistry
of Nucleotides?
•NDPs and NTPs are polyprotic acids (多質子酸)
10.3 What Is the Structure and Chemistry
of Nucleotides?
Cyclic nucleotides are cyclic phosphodiesters
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 10.13 Formation of ADP and ATP by the successive
addition of phosphate groups via phosphoric anhydride (磷
酸酐) linkages. Note that the reaction is a dehydration
synthesis.
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.
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. Nucleotidyl group transfer
is shown here.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
• Nucleoside 5’-triphosphates are indispensable
agents in metabolism because their phosphoric
anhydride (磷酸酐) bonds are a source of
chemical energy
• 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 protein synthesis
• 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. Pyrophosphoryl group
transfer is shown 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
•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 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.
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 RNA - the basis of structure and
function of ribosomes
• messenger RNA - carries the message for
protein synthesis
• transfer RNA - carries the amino acids for
protein synthesis
10.4 What Are Nucleic Acids?
Figure 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.
10.5 What Are the Different Classes of
Nucleic Acids?
Figure 10.16 The
antiparallel nature of the
DNA double helix.
• small nuclear RNA: gene splicing
• Others:
• small RNAs: 21~28 nucleotides, gene regulation
The DNA Double Helix
The double helix is stabilized by hydrogen bonds
• "Base pairs" arise from hydrogen bonds
• Erwin Chargaff had the pairing data, but didn't
understand its implications
• Rosalind Franklin's X-ray diffraction data of
DNA 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.
The Structure of DNA
The Base Pairs Postulated by Watson
Figure 10.17 The Watson-Crick base pairs A:T and G:C.
The Structure of DNA
An antiparallel double helix
Figure 10.18 Replication of DNA
gives identical progeny molecules
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
• 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 (組織
蛋白) proteins to form nucleosomes (核小體)
• Base pairs: A-T, G-C
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
Figure 10.20a 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.
Figure 10.19 The chromosome is shown surrounding the cell.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
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 – 100~200 residues stability of
mRNA
• Splicing produces final mRNA without introns
heterogeneous nuclear RNA
snRNA+ proteins = snRNPs
Figure 10.20b Transcription and translation of mRNA
molecules in prokaryotic versus eukaryotic cells.
In eukaryotes, a single mRNA codes for just one protein, but
structure is composed of introns and exons.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
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
S
Figure 10-23 Unusual bases in RNA.
Figure 10.22 The organization and composition of ribosomes.
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-a.a.
• Aminoacyl tRNA molecules are the
substrates of protein synthesis
The RNA World and Early Evolution
• Thomas Cech and Sidney Altman showed that RNA
molecules are not only informational – they can also be
catalytic
• This gave evidence to the postulate by Francis Crick and
others that prebiotic evolution (that is, early evolution
before cells arose) depended on self-replicating, 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
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 basepaired nucleotides in the
sequence.
The RNA World and Early Evolution
Aminoimidazolecarbonitrile is tetramer of HCN and
may be a precursor of adenine (a pentamer of HCN ).
The Chemical Differences Between DNA
and RNA Have Biological Significance
• Two fundamental chemical differences distinguish
DNA from RNA:
• DNA contains 2-deoxyribose instead of ribose
• DNA contains thymine instead of uracil
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
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 by using thymine
(5-methyl-U) in place of uracil
Figure 10.25 Deamination of cytosine forms uracil.
The Chemical Differences Between DNA
and RNA Have Biological Significance
DNA & RNA Differences?
Why is DNA 2'-deoxy and RNA is not?
Figure 10.26 The 5-methyl
group on thymine labels it
as a special kind of uracil.
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.
• 2'-OH group in RNA make the 3'-phosphodiester
bond of RNA more susceptible to alkaline
hydrolysis
• 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?
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attach 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?
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
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?
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?
The Enzymes that hydrolyze nucleic acids are phosphodiesterases.
Figure 10.28 Cleavage in polynucleotide chains.
Figure 10.28ab Cleavage on the a side leaves the phosphate
attached to the 5'-position of the adjacent nucleotide. b-side
hydrolysis yields 3'-phosphate products.
Restriction Enzymes
Type II Restriction Enzymes
• Bacteria have learned to "restrict" the
possibility of attack from foreign DNA by
means of "restriction 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"
• 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
• Names use 3-letter italicized code:
• 1st letter - genus; 2nd,3rd - species
• Following letter denotes strain
• EcoRI is the first restriction enzyme found in the
R strain of E. coli
Cleavage Sequences of Restriction
Endonucleases
Restriction Mapping of DNA
Figure 10.29
Restriction
mapping analysis.