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Nucleic Acids
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
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
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)
Heterocycles in DNA and RNA
• 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).
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
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
• 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
• 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.
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
• 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
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
• 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
• 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
• 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
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
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
• 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
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
Analytical Methods
• 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)
•
•
The solution also contains small amounts of the four 2,3-dideoxyribonucleoside
triphosphates (ddNTPs)
Each is modified with a different fluorescent dye molecule
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
• Step 1: The 5 –OH group on the sugar is protected as its p-dimethoxytrityl
(DMT) ether
DNA Synthesis: Protection
• Step 2: 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
DNA Synthesis: DMT Removal
• Step 2 (Continued): Removal of the DMT protecting group by
treatment with a moderately weak acid
DNA Synthesis: Coupling
• 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
• Phosphite is oxidized to phosphate by I2
• The cycle is repeated until the sequence is complete
DNA Synthesis- Step 5: Clean-up
• All protecting groups are removed and the product is released
from the support by treatment with aqueous NH3
The Polymerase Chain Reaction (PCR)
• 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
• 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
• 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