John E.

McMurry

http://www.cengage.com/chemistry/mcmurry

Chapter 24
Biomolecules: Nucleic Acids
and Their Metabolism

Richard Morrison University of Georgia, Athens

Nucleic Acids
Nucleic Acids are the chemical carriers of a cells genetic
information
Deoxyribonucleic acid (DNA)
Holds the information that determines the nature of a cell
Controls cell growth and division
Directs biosynthesis of the enzymes and other proteins
required for cellular functions
Ribonucleic acid (RNA)
Nucleic acid derivatives such as ATP are involved as
phosphorylating agents in many biochemical pathways
Several important coenzymes, including NAD+, FAD, and
coenzyme A, have nucleic acid components
24.1 Nucleotides and Nucleic Acids
Nucleic acids are biopolymers
Composed of nucleotides which are joined together to form a long chain
Nucleotide
Composed of nucleosides bound to a phosphate group
Nucleoside
Composed of an aldopentose sugar linked through its anomeric
carbon to the nitrogen atom of a heterocyclic purine or pyrimidine
base
Nucleotides and Nucleic Acids
DNA
Sugar component is 2-deoxyribose (the prefix 2-deoxy
indicates that oxygen is missing from the 2 position of
ribose)
Contains four different amino bases
Two substituted purines (adenine and guanine)
Two substituted pyrimidines (cytosine and thymine)
RNA
Sugar component is ribose
Contains adenine, guanine, and cytosine
Thymine is replaced by a closely related pyrimidine base

called uracil
Nucleotides and Nucleic Acids
The pyrimidines and purines found in DNA and RNA
Nucleotides and Nucleic Acids
Structures of the four deoxyribonucleotides
Nucleotides and Nucleic Acids
Structures of the four ribonucleotides
Nucleotides and Nucleic Acids
In naming and numbering nucleotides, positions on
the sugars are given a prime superscript to distinguish
them from positions on the amine base
DNA and RNA differ dramatically in size
Molecules of DNA have molecular weights up to 75
billion
Molecules of RNA are much smaller, containing as few
as 21 nucleotides, and have a molecular weight as low
as 7,000
Nucleotides and Nucleic Acids
Nucleotides are linked together in DNA and RNA by phosphodiester
bonds between the phosphate group at C5 on one nucleotide and the
3-hydroxyl group of the sugar of another nucleotide
C3 is one free hydroxyl group at the end of the nucleic
polymer (the 3 end)
C5 is another free hydroxyl group at the other end of the
nucleic polymer (the 5 end)
Sequence of nucleotides in a chain is described by starting at the 5 end
and identifying the bases in order of occurrence (using G, C, A, T or U)
24.2 Base Pairing in DNA: The Watson-
Crick Model
Samples of DNA isolated from different tissues of the
same species have the same proportions of
heterocyclic bases
Samples of DNA from different species often have greatly
different proportions of bases
Composition of human DNA
30% each of adenine and thymine
20% each of guanine and cytosine
Composition of the bacterium Clostridium perfringens
37% each of adenine and thymine
13% each of guanine and cytosine
Base Pairing in DNA: The Watson-Crick Model
In 1953, James Watson and Francis Crick proposed the secondary
structure of DNA
DNA under physiological conditions consists of two polynucleotide
strands
Strands run in opposite directions and coil around each other
in a double helix
The helix is 20 wide
The two strands are
complementary and are
held together by hydrogen
bonds between specific
pairs of bases
A with T

C with G
Base Pairing in DNA: The Watson-Crick Model
There are 10 base pairs per turn
Each turn is 34 in length
The two strands of the double helix coil in such a way that two
kinds of grooves result
A major groove 12 wide
A minor groove 6 wide
The grooves are lined with
hydrogen bond donors and
acceptors
A variety of flat, polycyclic
aromatic molecules are able
to slip sideways, or intercalate,
between the stacked bases
An organisms genetic
information is stored as a
sequence of deoxyribonucleotides strung together in the DNA chain
Base Pairing in DNA: The Watson-Crick Model
Central dogma of molecular genetics
The function of DNA is to store information and pass it to RNA
The function of RNA is to read, decode, and use the information
received from DNA to make proteins
Three fundamental processes take place:
Replication process by which identical copies of DNA are made so
the information can be preserved and handed down to offspring
Transcription the process by which the genetic messages are read
and carried out of the cell nucleus to ribosomes, where protein
synthesis occurs
Translation the process by which the genetic messages are
decoded and used to synthesize proteins
Worked Example 24.1
Predicting the Complementary Base Sequence in
Double-Stranded DNA
What sequence of bases on one strand of DNA is
complementary to the sequence TATGCAT on another
strand?
Worked Example 24.1
Predicting the Complementary Base Sequence in
Double-Stranded DNA

Strategy
Remember that A and G form complementary pairs with T
and C
Go through the sequence replacing A by T, G by C, T by A,
and C by G
Remember that the 5 end is on the left and the 3 end is on
the right in the original strand
Worked Example 24.1
Predicting the Complementary Base Sequence in
Double-Stranded DNA

Solution
Original: (5) TATGCAT (3)
Compliment: (3) ATACGTA (5) or
(5) ATGCATA (3)
24.3 Replication of DNA
Replication
An enzyme-catalyzed process
1. Begins with a partial unwinding of the double helix
2. Bases become exposed
3. New nucleotides line up on each strand in a complementary
manner (A with T and C with G)
4. Two new strands begin to grow
Each new strand is complementary to its old template strand
5. Two identical DNA helices are produced
The process is described as semiconservative replication
because each of the new DNA molecules contains one old
strand and one new strand
Replication of DNA
A representation of semiconservative DNA replication
Replication of DNA
Addition of nucleotides to the growing chain
Takes place in the 53 direction
Catalyzed by DNA polymerase
Key step is the addition of a nucleoside 5-triphosphate to
the free 3-hydroxyl group of the growing chain, with loss of
a diphosphate leaving group
Replication of DNA
Both new strands are synthesized in the 53 direction
They cannot be made in exactly the same way
One strand must have its 3 end near the point of unraveling (the
replication fork), while the other strand has its 5 end near the
replication fork
The complement of the original 53 strand is
synthesized continuously in a single piece
The compliment of the original 35 strand is
synthesized discontinuously in small pieces that are
often then linked by DNA ligases
24.4 Transcription of DNA
RNA
Similar to DNA but contains ribose instead of
deoxyribose and uracil instead of thymine
Three primary kinds
Messenger RNA (mRNA) carries genetic messages from
DNA to ribosomes,
Small granular particles in the cytoplasm of a cell where
protein synthesis takes place
Ribosomal RNA (rRNA) complexed with protein provides
the physical makeup of the ribosomes
Transfer RNA (tRNA) transports amino acids to the
ribosomes where they are joined together to make
proteins
Transcription of DNA
Genetic information in DNA is contained in segments
called genes
Each gene consists of a specific nucleotide sequence that
encodes a specific protein
Conversion of DNA information into proteins begins with
transcription of DNA to mRNA
Transcription of DNA
Promoter site
A specific base sequence found within a DNA chain
typically consisting of around 40 base pairs located
upstream (5) of the transcription start site
Consists of two hexameric consensus sequences, one
located 10 base pairs upstream and the second located
35 base pairs upstream from the beginning of the
coding region
Signals the beginning of a gene
Other base sequences signal a stop near the end of the
gene
Transcription of DNA
Transcription
The process by which genetic information encoded in DNA is read and
used to synthesize RNA in the nucleus of the cell
1. Several turns of the DNA double helix unwind, forming a bubble
and exposing the bases of the two strands
2. Ribonucleotides line up in the proper order by hydrogen bonding to
their complementary bases on DNA
3. Bond formation occurs in the 5 3 direction
4. The growing RNA molecule unwinds from DNA
Transcription of DNA
Only one of the two DNA strands is transcribed into
mRNA
The strand that contains the gene is called the coding
strand, or sense strand
The strand that gets transcribed is called the template
strand, or antisense strand
The RNA molecule produced during transcription is the
complement of the DNA antisense strand and is
therefore a copy of the DNA coding strand (except T
has been replaced with U)
Transcription of DNA
Genes are not continuous segments of the DNA
chain
1. A gene begins in an exon, a small section of DNA
2. Genes are interrupted by noncoding sections called
introns
3. Genes take up again further down the chain in another
exon
The final mRNA molecule results after the noncoded sections
are cut out and the remaining pieces are spliced together
90% of human DNA seems to be made up of introns
10% of DNA contains coding instructions
24.5 Translation of RNA: Protein Biosynthesis
Primary cellular function of mRNA
Direct biosynthesis of the thousands of diverse peptides and proteins
required by an organism
The mechanics of protein biosynthesis take place on ribosomes,
small granular particles in the cytoplasm of a cell that consist of
about 60% ribosomal RNA and 40% protein
The specific ribonucleotide sequence in mRNA forms a codon
that determines the order in which amino acid residues are joined
Each codon consists of a sequence of three

ribonucleotides that is specific for a given amino acid

The series UUC on mRNA is a codon directing incorporation
of the amino acid phenylalanine into the growing protein
64 possible triplets of the four bases in RNA

61 code for specific amino acids

3 code for chain termination
Translation of RNA: Protein Biosynthesis
Translation of RNA: Protein Biosynthesis
Translation
The process by which the genetic information transcribed
from DNA onto mRNA is read by tRNA and used to direct
protein synthesis
There are 61 different tRNAs, one for each of the 61
codons that specifies an amino acid
A typical tRNA is single-stranded and cloverleaf-shaped
On the middle leaf it contains an anticodon, a
sequence of three ribonucleotides complementary
to the codon sequence
Contains about 70 to 100 ribonucleotides

Bonded to a specific amino acid by an ester linkage

through the 3 hydroxyl on ribose at the 3 end of the
tRNA
Translation of RNA: Protein Biosynthesis
The codon sequence UUC present on mRNA is read by a
phenylalanine-bearing tRNA having the complementary
anticodon base sequence GAA
Nucleotide sequences are written in the 53 direction so the
sequence in an anticodon must be reversed
Translation of RNA: Protein Biosynthesis
1. Successive codons
on mRNA are read
2. Different tRNAs
bring the correct
amino acids into
position for enzyme-
mediated transfer to
the growing peptide
3. When synthesis of
the proper protein is
completed, a stop
codon signals the
end
4. The protein is
released from the
ribosome
Worked Example 24.2
Predicting the Amino Acid Sequence Transcribed
from DNA
What amino acid sequence is coded by the following segment
of a DNA coding strand?

(5) CTA-ACT-AGC-GGG-TCG-CCG (3)

Worked Example 24.2
Predicting the Amino Acid Sequence Transcribed
from DNA
Strategy
The mRNA produced during translation is a copy of the DNA
coding strand
Each T replaced by U
The mRNA has the sequence
(5) CUA-ACU-AGC-GGG-UCG-CCG (3)
Worked Example 24.2
Predicting the Amino Acid Sequence Transcribed
from DNA
Solution
Leu-Thr-Ser-Gly-Ser-Pro
24.6 DNA Sequencing
Methods for sequencing immense DNA chains
1. First step in sequencing
Cleave the DNA chain at known points to produce smaller
pieces, done through the use of restriction endonucleases
More than 3500 restriction enzymes are known
About 200 restriction enzymes are commercially available
Each different restriction enzyme cleaves a DNA molecule at
a point in the chain where a specific base sequence occurs
The restriction enzyme AluI cleaves between G and C in
the four-base sequence AG-CT
(5-AGCT-(3) sequence is that same as its complement (3)-
TCGA-(5) when both are read in the same 53 direction
DNA Sequencing
Two methods of DNA sequencing are available
The Maxam-Gilbert method
Uses chemical techniques
Sanger dideoxy method
Uses enzymatic reactions
The more commonly used of the two
Method responsible for sequencing the entire human
genome of 2.9 billion base pairs
In commercial sequencing instruments, the dideoxy
method begins with a mixture of the following:
The restriction fragment to be sequenced
DNA Sequencing
A small piece of DNA called a primer, whose sequence is
complementary to that on the 3 end of the restriction fragment
The four 2-deoxyribonucleoside triphosphates (dNTPs)
Very small amounts of the four 2 ,3-dideoxyribonucleoside
triphosphates (ddNTPs), each of which is labeled with a
fluorescent dye of a different color
(A 2 ,3 -dideoxyribonucleoside triphosphate in one in which
both 2 and 3 OH groups are missing from ribose)
DNA Sequencing
2. DNA polymerase is added to the mixture
A strand of DNA complementary to the restriction fragment begins
to grow from the end of the primer
Most of the time only normal deoxyribonucleotides are
incorporated into the growing chain
Sometimes a dideoxyribonucleotide is incorporated
When this occurs, DNA synthesis stops because the chain
end no longer has a 3 hydroxyl group for adding further
nucleotides
3. The product
Consists of a mixture of DNA fragments of all possible lengths, each
terminated by one of the four dye-labeled dideoxyribonucleotides
Mixture is then separated according to the size of the pieces by gel
electrophoresis
DNA Sequencing
The identity of the terminal dideoxyribonucleotide in each
piece and thus the sequence of the restriction fragment is
identified by noting the color with which it fluoresces
24.7 DNA Synthesis
Synthesis of short DNA segments, called oligonucleotides or
oligos
A nucleotide has multiple reactive sites that must be selectively
protected and deprotected at the proper times
Coupling of the four nucleotides must be carried out in the proper
sequence
Automated DNA synthesizers allow the fast and reliable synthesis
of DNA segments up to 200 nucleotides in length
A protected nucleotide is covalently bonded to a solid support
One nucleotide at a time is added to the growing chain by the use
of a coupling reagent
After the final nucleotide has been added, all the protecting
groups are removed and the synthetic DNA is cleaved from the
solid support
DNA Synthesis
Step 1 Attachment of a protected deoxynucleoside to a silica (SiO2)
support
Done through an ester linkage to the 3 OH group of the
deoxynucleoside
Both the 5 OH group on the sugar and free NH2 groups on the
heterocyclic bases must be protected
The deoxyribose 5 OH is protected as its p-dimethoxytrityl (DMT)
ether
DNA Synthesis
Adenine and cytosine bases are protected by benzoyl groups
Guanine is protected by an isobutryl group
Thymine requires no protection
DNA Synthesis
Step 2 Removal of the DMT protecting group by treatment
with dichloroacetic acid in CH2Cl2
Reaction occurs by an SN1 mechanism
Reaction proceeds rapidly due to the stability of the tertiary,
benzylic dimethoxytrityl cation
DNA Synthesis
Step 3 Coupling of the polymer-bonded deoxynucleoside
with a protected deoxynucleoside containing a
phosphoramidite group, R2NP(OR)2, at the 3 position
Takes place in the polar aprotic solvent acetonitrile
Requires catalysis by the heterocyclic amine tetrazole
Yields a phosphite, P(OR)3
DNA Synthesis
Step 4 Oxidation
Phosphite product is oxidized to a phosphate by treatment with iodine in
aqueous tetrahydrofuran in the presence of 2,6-dimethylpyridine
The cycle is repeated until oligonucleotide chain of the desired
sequence is built
1. Deprotection
2. Coupling
3. Oxidation
DNA Synthesis
Step 5 Final step
Removal of all
protecting groups
Cleavage of the ester
bond holding the DNA
to the silica
All reactions are
done at the same
time by treatment
with aqueous NH3
Purification by
electrophoresis yields
the synthetic DNA
24.8 The Polymerase Chain Reaction
Polymerase chain reaction (PCR)
A method for amplifying small amounts of DNA to
produce larger amounts
Invented by Kary Mullis in 1986
PCR produces multiple copies of a given DNA
sequence
Makes it possible to obtain several micrograms (1 ug
= 10-6 g; about 1011 nucleotides) in a few hours when
starting from less than 1 picogram of DNA with a
chain length of 10,000 nucleotides (1 pg = 10-12 g;
about 100,000 molecules)
The Polymerase Chain Reaction
Taq polymerase
The key to the polymerase chain reaction
A heat-stable enzyme isolated from the thermophilic
bacterium Thermus aquaticus found in a hot spring in
Yellowstone National Park
Able to take a single strand of DNA that has a short,
primer segment of complementary chain at one end
and then finish constructing the entire complementary
strand
Overall process takes three steps
The Polymerase Chain Reaction
The polymerase chain reaction
The Polymerase Chain Reaction
Step 1 Denaturation of the double-stranded DNA
The double-stranded DNA is heated in the presence
of:
Taq polymerase
Mg2+ ion
The four deoxynucleotide triphosphate monomers
(dNTPs)
A large excess of two short oligonucleotide primers of
about 20 bases each
Each primer is complementary to the sequence at the
end of one of the target DNA segments
Double-stranded DNA denatures at a temperature of
95 C, spontaneously breaking apart into two single
strands
The Polymerase Chain Reaction
Step 2
The temperature is lowered
Between 37 and 50 C
Allows primers to anneal by hydrogen bonding to their
complementary sequence at the end of each target strand
Step 3
Temperature is raised to 72 C
Taq polymerase catalyzes the addition of further nucleotides to the
two primed DNA strands
When replication is finished, two copies of the original DNA exist
Automated PCR
30 or so cycles can be carried out in an hour resulting in a theoretical
amplification factor of 230 (~109)
The efficiency of each cycle is less than 100%

Experimental amplification is about 106 to 108

24.9 Catabolism of Nucleotides
Dietary nucleic acids
First pass through the stomach to the intestines
Hydrolyzed to their constituent nucleotides by a variety of
different nucleases
Dephosphorylation by various nucleotidases gives nucleosides
A third cleavage by nucleosidases gives the constituent bases
Bases are catabolized to produce intermediates that enter other
metabolic processes
Catabolism of Nucleotides
Purine Catabolism: Guanosine
Guanosine and deoxyguanosine are both catalyzed
by a three-step pathway
1. Begins with cleavage to give guanine
2. Guanine is hydrolyzed to yield xanthine
3. Oxidation of xanthine gives uric acid, which is excreted
in the urine
Catabolism of Nucleotides
Figure 24.10
Pathway for the catabolism of guanosine and deoxyguanosine
to uric acid
Catabolism of Nucleotides
STEP 1 OF FIGURE 24.10: PHOSPHOROLYSIS
Phosphorolysis of guanosine (or deoxyguanosine)
Catalyzed by purine nucleoside phosphorylase
Gives b-ribose 1-phosphate (or b-deoxyribose 1-phosphate)
plus guanine
Reaction probably occurs by an SN1-like replacement of
guanine by phosphate ion through an oxonium-ion
intermediate
Catabolism of Nucleotides
STEP 2 OF FIGURE 24.10: HYDROLYSIS
Hydrolysis of guanine
Gives xanthine
Catalyzed by guanine deaminase
Occurs by nucleophilic addition of water to the C=N bond
followed by expulsion of ammonium ion
Catabolism of Nucleotides
STEP 3 OF FIGURE 24.10: OXIDATION
Xanthine
Oxidized by xanthine oxidase
A complex enzyme that contains FAD and an oxo-
molybdenum(VI) cofactor
1. A base deprotonates the Mo-OH group
2. The resulting anion does a nucleophilic addition to the C=N
bond in xanthine
3. The nitrogen anion expels hydride ion
4. Hydride ion adds to an Mo=S bond, thereby reducing the
molybdenum center from Mo(VI) to Mo(IV)
5. Hydrolysis of the Mo-O bond gives an enol
6. Enol tautomerizes to uric acid
7. The reduced molybdenum is reoxidized by O2 in a complex
redox pathway
Catabolism of Nucleotides
The mechanism of step 3 in Figure 24.10, oxidation of xanthine
to yield uric acid
Catabolism of Nucleotides
Adenosine (purine nucleotide)
Steps are similar to those for guanosine but order of
steps differs
Base in adenosine is first degraded and then
removed
24.10 Biosynthesis of Nucleotides
Purine Biosynthesis: Adenosine Monophosphate and
Guanosine Monophosphate
Purine nucleotides are formed by the initial attachment of an
NH2 group to the ribose, followed by multistep buildup of the
heterocyclic base
-NH2 attaches by a nucleophilic substitution reaction of
ammonia
Inosine monophosphate (IMP) is the first fully formed purine
ribonucleotide
Adenosine monophosphate (AMP) derived from IMP
Biosynthesis of Nucleotides
Adenosine monophosphate
Biosynthesized from IMP
AMP is biosynthesized in a three-step sequence:
1. Initial phosphorylation with GTP to form an imino
phosphate
Nucleophilic acyl substitution reaction
2. Reaction with aspartate to give adenylosuccinate
3. Elimination of fumarate
E1cB reaction
Biosynthesis of Nucleotides
Pathway for the conversion of inosine monophosphate to adenosine
monophosphate