Download Chapter 28. Heterocycles and Nucleic Acids

John E. McMurry
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Chapter 28
Biomolecules: Nucleic Acids
Paul D. Adams • University of Arkansas
Nucleic Acids
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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
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
28.1 Nucleotides and Nucleic
Acids
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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)
Heterocycles in DNA and RNA
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Adenine, guanine, cytosine and thymine are in DNA
RNA contains uracil rather than thymine
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)
Nucleotides (Continued)
Nucleotides (Continued)

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).
28.2 Base Pairing in DNA: The
Watson–Crick Model
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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
Hydrogen Bonds in DNA


The G-C base pair involves three H-bonds
The A-T base pair involves two H-bonds
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
Grooves
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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.
Nucleic Acids and Heredity
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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.
28.3 Replication of DNA


Begins with a partial unwinding of the double helix,
exposing the recognition site on the bases
When activated forms of the complementary nucleotides
(A with T and G with C) associate, two new strands begin
to grow
The Replication Process
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
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
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
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
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
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
Transcription Process
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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,
Transcription of RNA from
DNA
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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)
Mechanism of Transcription
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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
resuming farther down the chain in another exon,
with an intron between that is removed from the
mRNA
28.5 Translation of RNA: Protein
Biosynthesis
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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
Codon Assignments of Base
Triplets
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
The Structure of tRNA
Processing Aminoacyl tRNA
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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
28.6 DNA Sequencing
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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)-TCGA-(5)
Other restriction enzymes produce other cuts permitting
partially overlapping sequences of small pieces to be
produced for analysis
Analytical Methods
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The Maxam–Gilbert method uses organic
chemistry to cleave phosphate linkages 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
The Sanger Dideoxy and
Nucleotides
The fragment to be sequenced is combined with:
A) A small piece of DNA (primer), having a sequence that is
complementary to that on the 3 end of the restriction fragment
B) The four 2-deoxyribonucleoside triphosphates (dNTPs)
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The solution also contains small amounts of the four 2,3dideoxyribonucleoside triphosphates (ddNTPs)
Each is modified with a different fluorescent dye molecule
Dideoxy Method - Analysis
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The product is a mixture of dideoxy-terminated DNA
fragments with fluorescent tags
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These are separated according to weight by
electrophoresis and identified by their specific
fluorescence
28.7 DNA Synthesis
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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
Step 1: The 5 –OH group on the sugar is protected as its pdimethoxytrityl (DMT) ether
DNA Synthesis: Protection
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Step 2: After the final nucleotide has been added, the
protecting groups are removed and the synthetic DNA is
cleaved from the solid support
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The bases are protected from reacting
DNA Synthesis: DMT Removal
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Step 2 (Continued): Removal of the DMT protecting
group by treatment with a moderately weak acid
DNA Synthesis: Coupling
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Step 3: The polymer-bound (protected) deoxynucleoside
reacts with a protected deoxynucleoside containing a
phosphoramidite group at its 3 position, catalyzed by
tetrazole, a reactive heterocycle
DNA Synthesis- Step 4: Oxidation
and Cycling
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Phosphite is oxidized to phosphate by I2
The cycle is repeated until the sequence is
complete
DNA Synthesis- Step 5:
Clean-up
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All protecting groups are removed and the product is
released from the support by treatment with aqueous NH3
28.8 The Polymerase Chain
Reaction (PCR)
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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
heat-stable (to survive the unwinding)
Each cycle doubles the amount of material
This is an exponential template-driven organic
synthesis
PCR: Heating and Reaction
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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
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

PCR: Growing More Chains
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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
Let’s Work a Problem
What amino acid sequence is coded for by the
following mRNA base sequence?
(5’) CUA-GAC-CGU-UCC-AAG-UGA (3’)
Answer
In order to answer this question effectively, one
must refer to Table 28.1, the codon assignments of
base triplets. With this reference the amino acid
sequence can easily be determined: Leu-Asp-ArgSer-Lys-Stop