Chapter 28.
Biomolecules: Nucleic Acids
Why this Chapter?
Last, but not least of the 4 major classes of
biomolecules to be introduced
To introduce chemical details of DNA sequencing and
synthesis
Nucleic acids
DNA and RNA are chemical carriers of a
cell’s genetic information
 Coded in a cell’s DNA is the information
that determines the nature of the cell,
controls cell growth, division
 Nucleic acid derivatives are involved as
phosphorylating agents in biochemical
pathways

2
28.1 Nucleotides and Nucleic Acids




Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the
chemical carriers of genetic information
Nucleic acids are biopolymers made of nucleotides, aldopentoses
linked to a purine or pyrimidine and a phosphate
RNA is derived from ribose
DNA is from 2-deoxyribose
◦ (the ' is used to refer to positions on the sugar portion of a
nucleotide)
3
Heterocycles in DNA and RNA


Adenine, guanine, cytosine and thymine are in DNA
RNA contains uracil rather than thymine
4
Nucleotides

In DNA and RNA the
heterocycle is bonded
to C1 of the sugar and
the phosphate is
bonded to C5 (and
connected to 3’ of the
next unit)
5
Nucleotides join together in DNA and RNA by
as phosphate between the 5’-on one nucleotide
and the 3 on another
One end of the nucleic acid polymer has a free hydroxyl
at C3 (the 3 end), and the other end has a phosphate at
C5 (the 5 end).
6
28.2 Base Pairing in DNA: The Watson–
Crick Model




In 1953 Watson and Crick noted that DNA consists of
two polynucleotide strands, running in opposite
directions and coiled around each other in a double
helix
Strands are held together by hydrogen bonds between
specific pairs of bases
Adenine (A) and thymine (T) form strong hydrogen
bonds to each other but not to C or G
Guanine (G) and cytosine (C) form strong hydrogen
bonds to each other but not to A or T
7
Hydrogen Bonds in DNA


The G-C base pair involves three H-bonds
The A-T base pair involves two H-bonds
8
The Difference in the Strands



The strands of DNA are complementary because
of H-bonding
Whenever a G occurs in one strand, a C occurs
opposite it in the other strand
When an A occurs in one strand, a T occurs in the
other
9
Grooves



The strands of the DNA
double helix create two
continuous grooves (major
and minor)
The sugar–phosphate
backbone runs along the
outside of the helix, and
the amine bases hydrogen
bond to one another on
the inside
The major groove is
slightly deeper than the
minor groove, and both
are lined by potential
hydrogen bond donors
and acceptors.
10
Nucleic Acids and Heredity
Processes in the transfer of genetic information:
 Replication: identical copies of DNA are made
 Transcription: genetic messages are read and carried out of the cell nucleus
to the ribosomes, where protein synthesis occurs.
 Translation: genetic messages are decoded to make proteins.

11
28.3 Replication of DNA


Begins with a partial unwinding of the double helix,
exposing the recognition site on the bases
Activated forms of the complementary nucleotides (A
with T and G with C) associate two new strands begin to
grow
12
The Replication Process
Addition takes place 5  3, catalyzed by DNA
polymerase
 Each nucleotide is joined as a 5-nucleoside triphosphate
that adds a nucleotide to the free 3-hydroxyl group of the
growing chain

13
28.4 Transcription of DNA



RNA contains ribose rather than deoxyribose and uracil
rather than thymine
There are three major kinds of RNA - each of which
serves a specific function
They are much smaller molecules than DNA and are
usually single-stranded
14
Messenger RNA (mRNA)


Its sequence is copied from genetic DNA
It travels to ribsosomes, small granular particles in the
cytoplasm of a cell where protein synthesis takes place
15
Ribosomal RNA (rRNA)



Ribosomes are a complex of proteins and rRNA
The synthesis of proteins from amino acids and ATP
occurs in the ribosome
The rRNA provides both structure and catalysis
16
Transfer RNA (tRNA)
Transports amino acids to the ribosomes where they
are joined together to make proteins
 There is a specific tRNA for each amino acid
 Recognition of the tRNA at the anti-codon
communicates which amino acid is attached

17
Transcription Process
Several turns of the DNA double helix unwind, exposing
the bases of the two strands
 Ribonucleotides line up in the proper order by hydrogen
bonding to their complementary bases on DNA
 Bonds form in the 5  3 direction,

18
Transcription of RNA from DNA




Only one of the two DNA strands is transcribed into
mRNA
The strand that contains the gene is the coding or sense
strand
The strand that gets transcribed is the template or
antisense strand
The RNA molecule produced during transcription is a
copy of the coding strand (with U in place of T)
19
Mechanism of Transcription



DNA contains promoter sites that are 10 to 35 base
pairs upstream from the beginning of the coding region
and signal the beginning of a gene
There are other base sequences near the end of the
gene that signal a stop
Genes are not necessarily continuous, beginning gene in
a section of DNA (an exon) and then resume farther
down the chain in another exon, with an intron
between that is removed from the mRNA
20
28.5 Translation of RNA: Protein
Biosynthesis




RNA directs biosynthesis of peptides and proteins
which is catalyzed by mRNA in ribosomes, where
mRNA acts as a template to pass on the genetic
information transcribed from DNA
The ribonucleotide sequence in mRNA forms a
message that determines the order in which different
amino acid residues are to be joined
Codons are sequences of three ribonucleotides that
specify a particular amino acid
For example, UUC on mRNA is a codon that directs
incorporation of phenylalanine into the growing protein
21
Codon Assignments of Base Triplets
22
The Parts of Transfer RNA
There are 61 different tRNAs, one for each of the 61
codons that specifies an amino acid
 tRNA has 70-100 ribonucleotides and is bonded to a
specific amino acid by an ester linkage through the 3
hydroxyl on ribose at the 3 end of the tRNA
 Each tRNA has a segment called an anticodon, a sequence
of three ribonucleotides complementary to the codon
sequence

23
The Structure of tRNA
24
Processing Aminoacyl tRNA
As each codon on mRNA is read, tRNAs bring amino acids
as esters for transfer to the growing peptide
 When synthesis of the proper protein is completed, a "stop"
codon signals the end and the protein is released from the
ribosome

25
28.6 DNA Sequencing
The order of the bases along DNA contains the genetic
inheritance.
 Determination of the sequence is based on chemical
reactions rather than physical analysis
 DNA is cleaved at specific sequences by restriction
endonucleases
 For example, the restriction enzyme AluI cleaves
between G and C in the four-base sequence AG-CT
Note that the sequence is identical to that of its
complement, (3)-TC-GA-(5)
 Other restriction enzymes produce other cuts
permitting partially overlapping sequences of small
pieces to be produced for analysis

26
Analytical Methods



The Maxam–Gilbert method uses organic chemistry to
cleave phosphate linkages at with specificity for the
adjoining heterocycle
The Sanger dideoxy method uses enzymatic reactions
The Sanger method is now widely used and automated,
even in the sequencing of genomes
27
The Sanger Dideoxy and Nucleotides
The fragment to be sequenced is combined with:
A) A small piece of DNA (primer), whose sequence is complementary to
that on the 3 end of the restriction fragment
B) The four 2-deoxyribonucleoside triphosphates (dNTPs)



The solution also contains small amounts of the four 2,3dideoxyribonucleoside triphosphates (ddNTPs)
Each is modified with a different fluorescent dye molecule
28
Dideoxy Method - Analysis


The product is a mixture of dideoxy-terminated DNA
fragments with fluorescent tags
These are separated according to weight by electrophoresis
and identified by their specific fluorescence
29
28.7 DNA Synthesis




DNA synthesizers use a solid-phase method starting with an attached,
protected nucleotide
Subsequent protected nucleotides are added and coupled
Attachment of a protected deoxynucleoside to a polymeric or silicate
support as an ester of the 3 OH group of the deoxynucleoside
The 5 OH group on the sugar is protected as its p-dimethoxytrityl
(DMT) ether

30
DNA Synthesis: Protection


After the final nucleotide has been added, the protecting
groups are removed and the synthetic DNA is cleaved from
the solid support
The bases are protected from reacting
31
DNA Synthesis: DMT Removal

Removal of the DMT protecting group by treatment with
a moderately weak acid
32
DNA Synthesis: Coupling

The polymer-bound (protected) deoxynucleoside reacts
with a protected deoxynucleoside containing a
phosphoramidite group at its 3 position, catalyzed by
tetrazole, a reactive heterocycle
33
DNA Synthesis: Oxidation and Cycling


Phosphite is oxidized to phosphate by I2
The cycle is repeated until the sequence is complete
34
DNA Synthesis: Clean-up

All protecting groups are removed and the product is
released from the support by treatment with aqueous NH3
35
28.8 The Polymerase Chain Reaction
Copies DNA molecules by unwinding the double helix
and copying each strand using enzymes
 The new double helices are unwound and copied again
 The enzyme is selected to be fast, accurate and heatstable (to survive the unwinding)
 Each cycle doubles the amount of material
 This is exponential template-driven organic synthesis

36
PCR: Heating and Reaction

The subject DNA is heated (to separate strands) with
◦ Taq polymerase (enyzme) and Mg2+
◦ Deoxynucleotide triphosphates
◦ Two, oligonucleotide primers, each complementary to
the sequence at the end of one of the target DNA
segments
37
PCR: Annealing and Growing
Temperature is reduced to 37 to 50°C,
allowing the primers to form H-bonds to
their complementary sequence at the end
of each target strand
PCR:Taq Polymerase
 The temperature is then raised to 72°C,
and Taq polymerase catalyzes the addition
of further nucleotides to the two primed
DNA strands

38
PCR: Growing More Chains



Repeating the denature–anneal–synthesize cycle a second
time yields four DNA copies, a third time yields eight
copies, in an exponential series.
PCR has been automated, and 30 or so cycles can be
carried out in an hour
See figure 28.9
39
40
What three components make up nucleotides?
1.
2.
3.
4.
5.
disaccharides, heterocyclic
aromatic amines, and
20%
phosphate ions
monosaccharides, heterocyclic
aromatic amines, and
phosphate ions
monosaccharides, heterocyclic
aliphatic amines, and phosphate
ions
disaccharides, heterocyclic
aliphatic amines, and phosphate
ions
monosaccharides, heterocyclic
aliphatic amines, and sulfate
ions
1
20%
2
20%
20%
3
4
20%
5
Select the best name for the molecule below:
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
guanine monophosphate
guanosine monophosphate
deoxyguanidine monophosphate
deoxyguanosine monophosphate
riboguanidine monophosphate
1
2
5
How many base pairs does it take to
complete one turn of DNA?
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
2
5
6
10
It depends on the
sequence of bases that
make up each turn.
1
2
5
What is the DNA complement to the following
sequence?
5’-CTGAATCGGA-3’
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
5'-TCCGATTCAG-3'
5'-AGGCTAAGTC-3'
5'-GACTTAGCCT-3'
5'-CTGAATCGGA-3'
5'-GAATCGGACT-3'
1
2
5
Which of the following is true concerning
replication?
1.
2.
3.
4.
5.
Addition of nucleotides to the
20%
growing chain takes place in the
3’ to 5’ direction.
The process is said to be
“conservative.”
The process is catalyzed by DNA
polymerase.
The key step is a nucleophilic
attack by the 5’ hydroxyl of
deoxyribose upon the γ
phosphate of a nucleoside
triphosphate.
All of these
1
20%
2
20%
20%
3
4
20%
5
The picture shown below demonstrates:
The picture shown demonstrates:
20%
1.
2.
3.
4.
5.
the replication fork.
the semiconservative nature of
replication.
the antiparallel nature of DNA.
how one strand must be made
discontinuously while the other can
be made continuously.
All of these
1
20%
2
20%
20%
3
4
20%
5
Which of the following are produced
by transcription?
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
messenger RNA
transfer RNA
ribosomal RNA
All of these
None of these
1
2
5
In the figure shown, the red DNA strand is the:
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
sense strand
template strand
coding strand
RNA-like strand
All of these
1
2
5
The codons that make up the genetic code are said
to be unambiguous. What does this mean?
1.
2.
3.
4.
5.
All 64 codons are specific for
a particular amino acid.
Each of the 64 codons codes
for a different amino acid.
Each of the codons that code
for amino acids is specific for
only one amino acid.
Each of the 64 codons can
code for more than one
amino acid.
None of these
20%
1
20%
2
20%
20%
3
4
20%
5
In the figure below, the part shown in red is the:
20%
1.
2.
3.
4.
5.
anticodon.
acceptor stem.
anticodon loop.
aminoacyl group.
None of these
1
20%
2
20%
20%
3
4
20%
5
What peptide sequence would be formed by the
DNA template strand shown below:
3’-CTA-ACG-GGG-CCC-GCC-5’
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
Asp-Pro-Cys-Arg-Gly
Asp-Cys-Pro-Gly-Arg
Asp-Cys-Pro-Arg-Gly
Cys-Pro-Arg-Gly-Arg
None of these
1
2
5
What are restriction endonucleases?
1.
2.
3.
4.
5.
enzymes that catalyze the
hydrolysis of phosphodiester bonds
20%
of DNA strands containing a
particular base sequence
enzymes that randomly catalyze the
phosphodiester bonds of DNA
strands
enzymes that catalyze the
disruption of base pairing along an
entire DNA strand
enzymes that prevent hydrolysis
from occurring on a strand of DNA
enzymes that prevent nucleic acids
from being cleaved
1
20%
2
20%
20%
3
4
20%
5
Which of the following is not required in the chain
termination method for DNA sequencing?
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
ddNTPs
dNTPs
DNA polymerase
radioactive sulfur
primer
1
2
5
In synthetic DNA synthesis, what is true
about the following reaction?
In synthetic DNA synthesis, what is true about the following reaction?
1.
This represents the first step of DNA synthesis.
2.
This represents the removal of a protection group so the
nucleotide can join with another.
3.
The removal of the DMT protection group occurs via an
SN2 mechanism.
4.
This reaction does not occur if the base is thymine.
5.
This represents the removal of a protection group so the
nucleotide can join with another; and the removal of the
DMT protection group occurs via an SN2 mechanism.
20%
1
20%
2
20%
20%
3
4
20%
5
Which of the following is not required for
polymerase chain reaction?
20%
1.
2.
3.
4.
5.
20%
20%
20%
3
4
20%
RNA polymerase
Taq polymerase or
another heat-stable
polymerase
target DNA
primers
dNTPs
1
2
5
Why was the discovery of Taq polymerase the key
to polymerase chain reaction?
1.
2.
3.
4.
5.
Taq polymerase is a faster DNA
polymerase than that found in
mammals.
Taq polymerase has a lower error
rate than other DNA polymerases.
Taq polymerase needs no primer.
Taq polymerase does not denature
at temperatures of over 90° C,
allowing for automated replication.
All of these
20%
1
20%
2
20%
20%
3
4
20%
5