NUCLEIC

ACIDS
 Topic Outline:
 History of Nucleic Acids
 Structure and Function
 Types of Nucleic Acids
1. DNA
2. RNA
 Central Dogma of Life
 Friedrich Miescher in
1869
 isolated what he called nuclein from the
nuclei of pus cells
 Richard Altmann in 1889

 Nuclein was shown to have acidic

properties, hence it became called nucleic
acid
 1920s

 the tetranucleotide hypothesis was

introduced
 The Tetranucleotide
hypothesis
 Up to 1940 researchers were convinced
that hydrolysis of nucleic acids yielded the
four bases in equal amounts.
 Nucleic acid was postulated to contain
one of each of the four nucleotides, the
tetranucleotide hypothesis.
 Takahashi (1932) proposed a structure of
nucleotide bases connected by
phosphodiester linkages.
The Tetranucleotide
hypothesis
adenine phosphate uracil

pentose
pentose

phosphate
phosphate

pentose pentose

cytosine phosphate guanine

 Astbury and Bell in 1938

 First X-ray diffraction pattern of DNA is

published.
 The pattern indicates a helical
structure, indicated periodicity.
X-ray diffraction of DNA
Wilkins & Franklin (1952): X-ray
crystallography
 Avery, MacLeod, and Mc
Carty in 1944
 demonstrate DNA could “transform”
cells.
 Supporters of the tetranucleotide
hypothesis did not believe nucleic acid
was variable enough to be a molecule
of heredity and store genetic
information.
DNA is Genetic Material
 Erwin Chargaff in late
1940s
 used paper chromatography for
separation of DNA hydrolysates.
 Amount of adenine is equal to amount
of thymine and amount of guanine is
equal to amount of cytosine.
 Hershey and Chase in
1952
 confirm DNA is a molecule of heredity.
The Hershey-Chase Experiment
The Hershey-Chase Experiment
 Watson and Crick in 1953

 determine the structure of DNA

Watson & Crick Base pairing
 Francis Crick in 1958
 proposes the “central dogma of molecular biology” .
 Kornberg purifies DNA polymerase I
1969
 Entire genetic code determined
Nucleic Acids

• Nucleic Acids are very long, thread-like

polymers, made up of a linear array of monomers
called nucleotides.

• Nucleic acids vary in size in nature

• tRNA molecules contain as few as 80 nucleotides

• Eukaryotic chromosomes contain as many as

100,000,000 nucleotides.
Two types of nucleic acid
are found
 Deoxyribonucleic acid (DNA)
 Ribonucleic acid (RNA)
 DNA and RNA
DNA
deoxyribonucleic acid
nucleic acid that stores genetic information
found in the nucleus of a mammalian cell.

RNA
ribonucleic acid
3 types of RNA in a cell
Ribosomal RNAs (rRNA) are components of ribosomes
Messenger RNAs (mRNA) carry genetic information
Transfer RNAs (tRNA) are adapter molecules in translation
The distribution of nucleic
acids in the eukaryotic
 DNA is found in the nucleus
cell
with small amounts in mitochondria and
chloroplasts
 RNA is found throughout the cell
The nucleus contains the cell’s DNA
(genome)
Nucleus
RNA is synthesized in the nucleus
and
exported to the cytoplasm
Nucleus

Cytoplasm
DNA as genetic material:
The circumstantial evidence
1. Present in all cells and virtually restricted to the nucleus
2. The amount of DNA in somatic cells (body cells) of any
given species is constant (like the number of
chromosomes)
3. The DNA content of gametes (sex cells) is half that of
somatic cells.
In cases of polyploidy (multiple sets of chromosomes)
the DNA content increases by a proportional factor
4. The mutagenic effect of UV light peaks at 253.7nm. The
peak for the absorption of UV light by DNA
NUCLEIC ACID STRUCTURE

 Nucleic acids are polynucleotides

 Their building blocks are nucleotides
NUCLEOTIDE STRUCTURE

PHOSPATE SUGAR BASE

Ribose or PURINES PYRIMIDINES
Deoxyribose
Adenine (A) Cytocine (C)
Guanine(G) Thymine (T)
Uracil (U)

NUCLEOTIDE
 Nucleotide Structure
All nucleotides contain three components:
1. A nitrogen heterocyclic base
2. A pentose sugar
3. A phosphate residue
Ribose is a pentose

C5

C4 C1

C3 C2
Spot the difference

RIBOSE DEOXYRIBOSE

CH2OH CH2OH
O OH O OH

C C C C

H H H H H H H H

C C C C

OH OH OH H
Chemical Structure of DNA vs RNA
Ribonucleotides have a 2’-OH
Deoxyribonucleotides have a 2’-H
 P

THE SUGAR-PHOSPHATE
BACKBONE P

 The nucleotides are all

orientated in the same P

direction
 The phosphate group joins the P

3rd Carbon of one sugar to the

5th Carbon of the next in line. P

P
 P
G

ADDING IN THE BASES

P
C

 The bases are

P
attached to the 1
st
C
Carbon
 Their order is P
A
important
It determines the P
genetic information of T
the molecule
P
T
 Hydrogen bonds

DNA IS MADE OF P
G
TWO STRANDS OF C
P
POLYNUCLEOTIDE P
C G
P
P
C G
P
P
A T
P
P
T A
P
P
T A
P
DNA IS MADE OF TWO STRANDS OF
POLYNUCLEOTIDE
 The sister strands of the DNA molecule run in opposite
directions (antiparallel)
 They are joined by the bases
 Each base is paired with a specific partner:
A is always paired with T
G is always paired with C
“Purine with Pyrimidine”
 The sister strands are complementary but not identical
 The bases are joined by hydrogen bonds, individually
weak but collectively strong
 There are 10 base pairs per turn
 Purines & Pyrimidines
Structure of Nucleotide
Bases
5’ End
Nucleotides
are
linked by
phosphodies
ter
bonds

3’ End
From DNA to Protein
DNA to Protein
 DNA acts as a “manager” in the process of
making proteins
 DNA is the template or starting sequence
that is copied into RNA that is then used
to make the protein
Central Dogma

 One gene – one protein

 Central Dogma

 This is the same for bacteria to humans

 DNA is the genetic instruction or gene
 DNA  RNA is called Transcription
 RNA chain is called a transcript
 RNA  Protein is called Translation
Expression of  Some genes are
transcribed in large
Genes quantities because
we need large
amount of this
protein
 Some genes are
transcribed in
small quantities
because we need
only a small
amount of this
protein
Nucleotides as
Language
We must start to think of the nucleotides – A,
G, C and T as part of a special language – the
language of genes that we will see translated
to the language of amino acids in proteins
Genes as Information Transfer

 A gene is the sequence of nucleotides within

a portion of DNA that codes for a peptide or a
functional RNA
 Sum of all genes = genome
STEP 1 – DNA REPLICATION
 DNA
Replication
 Semiconservative
 Daughter DNA is a
double helix with 1
parent strand and 1 new
strand
 Found that 1 strand
serves as the template
for new strand
DNA Template

 Each strand of the parent DNA is used as a template

to make the new daughter strand
 DNA replication makes 2 new complete double
helices each with 1 old and 1 new strand
 Replication Origin
 Site where replication
begins
 1 in E. coli
 1,000s in human
 Strands are separated to
allow replication machinery
contact with the DNA
 Many A-T base pairs because
easier to break 2 H-bonds that
3 H-bonds
 Note anti-parallel chains
 Replication Fork

 Bidirectional movement of the DNA replication machinery

THE REPLICATION FACTORY

 DNA replication is an intricate process

requiring the concerted action of many
different proteins.

 The replication proteins are clustered

together in particular locations in the cell and
may therefore be regarded as a small
“Replication Factory” that manufactures DNA
copies.
THE REPLICATION FACTORY

 The DNA to be copied is fed through the factory,

much as a reel of film is fed through a movie
projector.

 The incoming DNA double helix is split into two

single strands and each original single strand
becomes half of a new DNA double helix.
Because each resulting DNA double helix retains
one strand of the original DNA, DNA replication
is said to be semi-conservative.
DNA REPLICATION PROTEINS

 DNA replication requires a variety of proteins.

 Each protein performs a specific function in

the production of the new DNA strands.

 Helicase, made of six proteins arranged in a

ring shape, unwinds the DNA double helix
into two individual strands.
 Single-strand binding proteins, or SSBs, are
tetramers that coat the single-stranded DNA.

 This prevents the DNA strands from reannealing

to form double-stranded DNA.

 Primase is an RNA polymerase that synthesizes

the short RNA primers needed to start the
strand replication process.
 DNA polymerase is a hand-shaped enzyme that strings
nucleotides together to form a DNA strand.

 The sliding clamp is an accessory protein that helps hold the

DNA polymerase onto the DNA strand during replication.

 RNAse H removes the RNA primers that previously began

the DNA strand synthesis.

 DNA ligase links short stretches of DNA together to create

one long continuous DNA strand.
Components of the DNA
Replication
Polymerase & Proteins
Coordinated

 One polymerase complex apparently synthesizes

leading/lagging strands simultaneously
 Even more complicated in eukaryotes
STRAND SEPARATION

 To begin the process of DNA replication, the two double

helix strands are unwound and separated from each
other by the helicase enzyme.

 The point where the DNA is separated into single

strands, and where new DNA will be synthesized, is
known as the replication fork.

 Single-strand binding proteins, or SSBs, quickly coat the

newly exposed single strands. SSBs maintain the
separated strands during DNA replication.
 Replication Fork

 Bidirectional movement of the DNA replication machinery

STRAND SEPARATION

 Without the SSBs, the complementary DNA strands

could easily snap back together.

 SSBs bind loosely to the DNA, and are displaced when

the polymerase enzymes begin synthesizing the new
DNA strands.
NEW STRAND SYNTHESIS

 Now that they are separated, the two single

DNA strands can act as templates for the
production of two new, complementary DNA
strands.

 Remember that the double helix consists of

two antiparallel DNA strands with
complementary 5’ to 3’ strands running in
opposite directions.
NEW STRAND SYNTHESIS

 Polymerase enzymes can synthesize nucleic

acid strands only in the 5’ to 3’ direction,
hooking the 5’ phosphate group of an incoming
nucleotide onto the 3’ hydroxyl group at the
end of the growing nucleic acid chain.

 Because the chain grows by extension off the 3’

hydroxyl group, strand synthesis is said to
proceed in a 5’ to 3’ direction.
 NEW STRAND SYNTHESIS
 Even when the strands are separated, however, DNA
polymerase cannot simply begin copying the DNA.

 DNA polymerase can only extend a nucleic acid chain but

cannot start one from scratch.

 To give the DNA polymerase a place to start, an RNA

polymerase called primase first copies a short stretch of the
DNA strand.

 This creates a complementary RNA segment, up to 60

nucleotides long that is called a primer.
 NEW STRAND SYNTHESIS
 Now DNA polymerase can copy the DNA strand.

 The DNA polymerase starts at the 3’ end of the RNA primer,

and, using the original DNA strand as a guide, begins to
synthesize a new complementary DNA strand.

 Two polymerase enzymes are required, one for each parental

DNA strand.

 Due to the antiparallel nature of the DNA strands, however,

the polymerase enzymes on the two strands start to move in
opposite directions.
 NEW STRAND SYNTHESIS
 One polymerase can remain on its DNA template
and copy the DNA in one continuous strand.

 However, the other polymerase can only copy a

short stretch of DNA before it runs into the primer
of the previously sequenced fragment.

 It is therefore forced to repeatedly release the DNA

strand and slide further upstream to begin
extension from another RNA primer.
 NEW STRAND SYNTHESIS
 The sliding clamp helps hold this DNA polymerase onto the
DNA as the DNA moves through the replication machinery.
The sliding clamp makes the polymerase processive.

 The continuously synthesized strand is known as the leading

strand, while the strand that is synthesized in short pieces is
known as the lagging strand.

 The short stretches of DNA that make up the lagging strand

are known as Okazaki fragments.
THE LAGGING STRAND

 Before the lagging-strand DNA exits the

replication factory, its RNA primers must be
removed and the Okazaki fragments must be
joined together to create a continuous DNA
strand.

 The first step is the removal of the RNA

primer.
THE LAGGING STRAND

 RNAse H, which recognizes RNA-DNA hybrid helices,

degrades the RNA by hydrolyzing its phosphodiester
bonds. Next, the sequence gap created by RNAse H is
then filled in by DNA polymerase which extends the 3’
end of the neighboring Okazaki fragment.

 Finally, the Okazaki fragments are joined together by

DNA ligase that hooks together the 3’ end of one
fragment to the 5’ phosphate group of the neighboring
fragment in an ATP- or NAD+-dependent reaction.
REPLICATION IN ACTION

 The process begins when the helicase

enzyme unwinds the double helix to expose
two single DNA strands and create two
replication forks.
 DNA replication takes place simultaneously
at each fork. The mechanism of replication is
identical at each fork.
 How is DNA Synthesized?

 Original theory
 Begin adding nucleotides at origin
 Add subsequent bases following pairing rules
 Expect both strands to be synthesized simultaneously
 This is NOT how it is accomplished
How is DNA Synthesized?
 Actually how DNA is synthesized
 Simple addition of nucleotides along one strand, as
expected
 Called the leading strand
 DNA polymerase reads 3’  5’ along the leading
strand from the RNA primer
 Synthesis proceeds 5’  3’ with respect to the new
daughter strand
 Remember how the nucleotides are added!!!!!
5’  3’
Mistakes during
Replication
 Base pairing rules must be maintained
 Mistake = genome mutation, may have consequence
on daughter cells
 Only correct pairings fit in the polymerase active
site
 If wrong nucleotide is included
 Polymerase uses its proofreading ability to cleave the
phosphodiester bond of improper nucleotide
 Activity 3’  5’
 And then adds correct nucleotide and proceeds down
the chain again in the 5’  3’ direction
Proofreading
DNA Repair

 For the rare mutations occurring during

replication that isn’t caught by DNA
polymerase proofreading
 For mutations occurring with daily assault
 If no repair
 In germ (sex) cells  inherited diseases
 In somatic (regular) cells  cancer
CONSEQUENCES OF GENETIC ERRORS
:SOURCES OF GENETIC VARIATION
 Mutation - any novel genetic change in the
gene complement or genotype relative to the
parental genotypes, beyond that achieved by
genetic recombination during meiosis.

 Mutations are changes in DNA structure, and

therefore changes in protein and phenotype.
CONSEQUENCES OF GENETIC ERRORS
SOURCES OF GENETIC VARIATION
 Mutations are rare! For every 100 million
nucleotides added to a developing DNA strand
only one mistake occurs on average.

 Mutations are heritable; and may be

beneficial, neutral, lethal, detrimental or
harmful to the organism.
Types of Mutation

1. Induced
 viruses, UV radiation, some chemicals
(nitric acid changes cytosine to uracil) or
mutagens (or carcinogens - benzene,
cigarette smoke).
Types of Mutation

2. Spontaneous
 Proofreading mistakes during DNA replication
(Base substitutions) - not necessarily a serious
change.

 Frame shift mutation (Addition or deletion of a

base) - serious change!
Types of Mutation

 A 3 letter code or codon is analogous to three letter words in a

sentence.
 Original sequence

THE CAT SAW THE DOG

 Base or letter substitutions

THE BAT SAW THE DOG
THE CAT SAW THE HOG
THE CAB SAW THE DOG
THE CAT SAW SHE DOG
THE CAT SAD THE DOG
THE CAT SAW THE DOC
Types of Mutation

 Deletions
THE CAT SAW TED OG
THE ATS AWT HED OG

 Additions
THE CAT SAW THE ZDO G
THE CMA TAS WTH EDO G
Types of Mutation

3. Jumping genes, transposable elements, or

transposons.
 Discovered by Barbara McClintok (1956)
while studying color variation in Indian
corn.
 Won Nobel prize in 1983.
Types of Mutation

3. Jumping genes, transposable elements, or transposons.

 Patches of yellow sometimes occur among the purple
grains of Indian corn. She explain this by assuming that
the gene was being interrupted by a foreign sequence
of DNA.
 These foreign bits of DNA could insert or remove
themselves from a stretch of DNA causing the genes
that they affected to be turned on or off. Such "jumping
genes" could copy themselves and move about within
the genome of the organism they occupied.
Types of Mutation

4. Chromosomal mutations (disruption in chromosomal

morphology - inversions and translocations).

5. Homeotic genes
 master genes that regulate suites of other genes and
may affect developmental pathways especially during
embryogenesis. Mutations in these master genes can
cause genetic anomalies. For example, a fruit fly that
possesses legs where antennae should be, or a
mosquito that has its mouth parts transformed into
legs.
Effect of Mutation
Uncorrected Replication
Errors

 Mismatch repair
 Enzyme complex recognizes mistake and excises newly-
synthesized strand and fills in the correct pairing
Mismatch Repair – cont’d

 Eukaryotes “label”
the daughter strand
with nicks to
recognize the new
strand
 Separates new from
old
Chemical Modifications
Thymine Dimers

 Caused by exposure to UV light

 2 adjacent thymine residues become
covalently linked
Repair
Mechanisms
 Different enzymes
recognize, excise
different mistakes
 DNA polymerase
synthesizes proper
strand
 DNA ligase joins new
fragment with the
polymer
STEP 2 - TRANSCRIPTION
Transcription
 The region of the double-stranded DNA
corresponding to a specific gene is copied
into an RNA molecule, called messenger RNA
(mRNA).
 RNA differs from DNA
 Ribose is the sugar rather than deoxyribose –
ribonucleotides
 U instead of T; A, G and C the same
 Single stranded
 Can fold into a variety of shapes that allows RNA to
have structural and catalytic functions
RNA
Differences
RNA Differences
Transcription
 Similarities to DNA replication
 Open and unwind a portion of the DNA
 1 strand of the DNA acts as a template
 Complementary base-pairing with DNA
 Differences
 RNA strand does not stay paired with DNA
 DNA re-coils and RNA is single stranded
 RNA is shorter than DNA
 RNA is several 1000 bp or shorter whereas DNA is
250 million bp long
 Catalyzes the formation
of the phosphodiester
RNA Polymerase
bonds between the
nucleotides (sugar to
phosphate)
 Uncoils the DNA, adds
the nucleotide one at a
time in the 5’ to 3’ fashion
 Uses the energy trapped
in the nucleotides
themselves to form the
new bonds
Template to Transcripts

 The RNA transcript is identical to the NON-

template strand with the exception of the T’s
becoming U’s
RNA Elongation

 Reads template 3’
to 5’
 Adds nucleotides
5’ to 3’ (5’
phosphate to 3’
hydroxyl)
 Synthesis is the
same as the
leading strand of
DNA
Differences in
DNA and RNA Polymerases
 RNA polymerase adds ribonucleotides not
deoxynucleotides
 RNA polymerase does not have the ability to
proofread what they transcribe
 RNA polymerase can work without a primer
 RNA will have an error 1 in every 10,000
nucleotides (DNA is 1 in 100,000,000 nucleotides)
 Types of RNA

 messenger RNA (mRNA) – codes for proteins

 ribosomal RNA (rRNA) – forms the core of the
ribosomes, machinery for making proteins
 transfer RNA (tRNA) – matches code for
amino acid on mRNA and positions the right
amino acid in place during protein synthesis
How does the process of
transcription begin?
 The DNA serves as the template for
producing an RNA transcript or copy of
information stored on the DNA molecule.

 The DNA molecule must open up and allow

an enzyme called RNA polymerase read and
connect together the sequence of nucleotides
in the proper order.
STEP 3 – TRANSLATION
RNA to Protein

 Translation is the process of turning

mRNA into protein
 Translate from one “language” (mRNA
nucleotides) to a second “language”
(amino acids)
 Genetic code – nucleotide sequence
that is translated to amino acids of the
protein
DNA Code

 Nucleotides read 3 at a time meaning that there

are 64 combinations for a codon (set of 3
nucleotides)
 Only 20 amino acids
 More than 1 codon per AA – degenerate code with the
exception of Met and Trp (least abundant AAs in
proteins)
Reading Frames

 Translation can occur in 1 of 3 possible reading

frames, dependent on where decoding starts in the
mRNA
Transfer RNA  Translation requires an
Molecules adaptor molecule that
recognizes the codon on
mRNA and at a distant
site carries the
appropriate amino acid
 Intra-strand base pairing
allows for this
characteristic shape
 Anticodon is opposite
from where the amino
acid is attached
Wobble Base
Pairing

 Due to degenerate code for amino acids some

tRNA can recognize several codons because the 3rd
spot can wobble or be mismatched
 Allows for there only being 31 tRNA for the 61
codons
Attachment of AA to tRNA

 Aminoacyl-tRNA synthase is the enzyme

responsible for linking the amino acid to the
tRNA
 A specific enzyme for each amino acid and
not for the tRNA
2 ‘Adaptors’ Translate
Genetic Code to Protein

2

1
Ribosomes  Complex machinery that
controls protein synthesis
 2 subunits
 1 large – catalyzes the peptide
bond formation
 1 small – binds mRNA and tRNA
 Contains protein and RNA
 rRNA central to the catalytic
activity
 Folded structure is highly conserved
 Protein has less homology and
may not be as important
 Ribosome Structures

 May be free in cytoplasm or attached to the ER

 Subunits made in the nucleus in the nucleolus and
transported to the cytoplasm
 Ribosomal Subunits

 1 large subunit – catalyzes the formation of the peptide bond

 1 small subunit – matches the tRNA to the mRNA
 Moves along the mRNA adding amino acids to growing protein
chain
Ribosomal Movement

E-site

 4 binding sites
 mRNA binding site
 Peptidyl-tRNA binding site (P-site)
 Holds tRNA attached to growing end of the peptide
 Aminoacyl-tRNA binding site (A-site)
 Holds the incoming AA
 Exit site (E-site)
Summary