Chapter 12: Molecular Genetics
Discovery of DNA as genetic material
• Mid-1900s - scientists knew the following about
chromosomes:
• They contained genetic information
• They were made up of DNA (deoxyribonucleic
acid) and proteins
• They did NOT know whether the DNA or the
protein was the actual genetic material.
• Several experiments were done to show that DNA
was the genetic material.
Frederick Griffith’s experiment – 1928
•Worked with 2 strains of bacteria
• S (smooth) strain caused pneumonia (coat
protects it from host’s immune system, and
host dies – top picture)
• R (rough) strain did not cause pneumonia (no
coat, is killed by host’s immune system –
bottom picture)
Results:
•Live S strain – mouse died
•Live R strain – mouse lived
•Heat-killed S strain – mouse lived
•Mixture of heat-killed S strain and live
R strain – mouse died
• When Griffith isolated live bacteria from
the dead mice who has been injected
with the mixture of heat-killed S strain
and live R strain, he found the smooth
trait.
• This suggested that the disease-causing
trait had been passed to the live R
bacteria.
• So live R bacteria were transformed into
live S bacteria – wondered what the
transforming substance was?
• Today, we know why this happened.
• The transforming substance was DNA.
• The heat killed the S strain bacteria,
but not its DNA.
• S strain DNA was taken up by live R
strain bacteria, allowing it to grow the
protective coat of the S strain.
• Transformed bacteria caused the mice
to die. 
Oswald Avery’s experiment – 1944
•Set out to identify transforming substance
from Griffith’s experiment
• Isolated different molecules (DNA,
proteins, lipids) from killed S cells
• Exposed live R cells to each molecule
separately
• When live cells exposed to DNA, they
transformed into S cells.
• Avery concluded that when S cells
were killed, their DNA was released
• R bacteria took that DNA in, and they
transformed into S cells
• Avery’s conclusions were not widely
accepted (although he was right!!)
Hershey and Chase experiment – 1952
•Alfred Hershey and Martha Chase showed
that DNA was the genetic material through
two experiments with a T2 (type 2) virus.
•They knew the following to be true:
• Viruses are made of DNA and protein
• Viruses inject genetic material into
bacterium to reproduce
•In first experiment, viral DNA was labeled
with radioactive phosphorous.
•Virus was allowed to inject genetic material
into bacterium.
•In second experiment, viral protein was
labeled with radioactive sulfur.
•Virus was allowed to inject genetic material
into bacterium.
•Because radioactive phosphorous was
found inside the bacteria, and radioactive
sulfur was not, they showed that DNA and
not protein was the genetic material found
in chromosomes.
Animation
Discovery of the structure of DNA
•The following was known about DNA by the early
1950’s:
•DNA is made of nucleotides
•Phosphate
•Sugar – deoxyribose
•1 of 4 bases – adenine, cytosine, thymine, and
guanine
•Chargaff’s rules –
•Amount of adenine and thymine always equal
•Amount of cytosine and guanine always equal
•DNA is in the shape of a double helix –
discovered by Franklin & Wilkins through
X-ray diffraction of DNA (a)
•1953 - Watson & Crick used above
information to construct 1st model of DNA
(b)
Structure of DNA
• DNA is a polynucleotide; nucleotides are
composed of:
• Phosphate
• Sugar (deoxyribose)
•1 of 4 nitrogencontaining bases adenine (A),
thymine (T),
guanine (G), and
cytosine (C)
•There are 2 strands of nucleotides
•2 strands are held together by hydrogen
bonds
•Two strands twist around each other to
form a double helix
•A & T, C & G are complementary base
pairs (purine to a pyrimidine)
•Purines – A & G
•Pyrimidines – T & C
•DNA strands are anti-parallel – run in
opposite directions – orientation of sugars
•5’ – pronounced 5 prime
•3’ – pronounced 3 prime
•When double
helix is unwound,
it resembles a
ladder
•A & T pair with 2
hydrogen bonds
•C & G pair with
3 hydrogen bonds
DNA Replication
• Purpose: DNA makes an exact copy of
itself prior to cell division; ensures that
each new cell gets a complete copy of the
DNA
DNA
DNA
Replication
DNA
DNA
Cell Division
DNA
• Steps: (enzymes in red)
1. Helicase attaches to DNA and breaks H2
bonds between bases – DNA chain
unwinds and unzips (Special proteins keep
it unzipped)
2. RNA primase adds an RNA primer (short
segment of RNA) to each strand of DNA
3. DNA polymerase attaches to separated
strand, helping add complementary
nucleotides to the new DNA strand
• Each side is done differently, since new
nucleotides can be added to the 3’ end of
the new strand only
• Leading strand – built continuously
• Lagging strand – elongates away from
elongation fork. Made in small sections
called Okazaki fragments
• Okazaki fragments are later connected
by the enzyme DNA ligase
Overview of DNA replication
Ladder configuration and DNA
replication
• Each old strand of nucleotides serves as a
template for each new strand.
• The process is semiconservative because each
new double helix is composed of an old strand of
nucleotides from the parent strand and one
newly-formed strand (daughter strand).
• Proofreading and repair limits error rate to less
than 1 per billion nucleotides.
Replication fork
DNA replication song
http://www.stolaf
.edu/people/gian
nini/flashanimat/
molgenetics/dnarna2.swf
DNA
RNA
(Deoxyribonucleic acid)
(Ribonucleic acid)
Sugar
Deoxyribose
Ribose
Bases
Adenine, uracil,
guanine, cytosine
Single-stranded
Helix
Adenine, thymine,
guanine, cytosine
Double-stranded
with base pairing
Yes
Location
Nucleus
Types
XXXXXXXXX
Nucleus,
cytoplasm
Messenger,
transfer, ribosomal
Strands
No
RNA
vs.
DNA
•Messenger RNA - carries genetic
information to the ribosomes
•Ribosomes - part of the cell where
proteins are made
•Ribosomal RNA - found in the
ribosomes
•Transfer RNA - transfers amino acids
to the ribosomes
Making Proteins
• A gene is a segment of DNA that specifies
the amino acid sequence of a protein.
• DNA is found in the nucleus of a cell;
proteins are made outside the nucleus at the
ribosomes.
Overview of gene expression
• Two processes are involved in the synthesis
of proteins in the cell:
• Transcription – DNA is copied into mRNA,
which will take a copy of the DNA code to
the ribosome to direct the making of
protein; occurs in nucleus
• Translation - the process of building
proteins, the sequence of bases of mRNA is
“translated” into a sequence of amino
acids; occurs in ribosome
• These processes are the same in all organisms
The Genetic Code
• DNA holds instructions to make a protein
• Instructions are copied into mRNA, which
will be used to make a protein
• Codon - each three-letter unit of an mRNA
molecule
• Each codon represents 1 amino acid
• There are 64 possible codons, and only 20
amino acids, so most amino acids have
more than one codon
Messenger RNA codons
•
•
•
•
•
Transcription
Purpose – Makes a copy of the DNA code
that can leave the nucleus and travel to
the ribosome to direct protein synthesis –
mRNA
Occurs in the nucleus
Occurs at only 1 gene at a time
Adenine in DNA pairs with uracil in
RNA, not thymine
Thymine in DNA pairs with adenine in
RNA
Steps:
1. Starting at promoter (signals the start of a gene),
segment of DNA unwinds and unzips
2. ½ of DNA will serve as a template (DNA template
strand is in the 3’ to 5’ direction; RNA in 5’ to 3’)
3. RNA polymerase joins the RNA nucleotides so
that the codons in mRNA are complementary to
the code in DNA.
4. Termination signal (signals end of gene) is
reached, process ends, and DNA closes back up
Animation
Animation
From video shown
http://www.youtube.com/watch?v=41_Ne5mS2ls
in class:
Transcription and mRNA synthesis
•
•
•
•
RNA Processing
DNA contains exons (parts of a gene that are
expressed) and introns (intragene segments –
not expressed)
Before mRNA leaves the nucleus, the introns
are removed so that only the exons remain
The splicing of mRNA is done by ribozymes,
enzymes composed of RNA.
Primary mRNA/pre-mRNA (with introns &
exons) is processed into mature mRNA
(without introns).
Translation
• Protein constructed during this process
• Occurs at the ribosomes
• Key players in translation:
• mRNA (messenger RNA)
• Made during transcription, has codons
• Travels from nucleus to ribosome
• Contains copy of DNA code to make
protein
• tRNA (transfer RNA)
• rRNA (ribosomal RNA)
Transfer RNA (tRNA)
• tRNA molecules bring amino acids to the
ribosomes
• Free-floating in the cytoplasm of the cell
•Each tRNA has a
sequence of nucleotides
called an anticodon – it is
this sequence that
determines which amino
acid each tRNA has
•Complementary base pairing occurs
between anticodons of tRNA and codons
of mRNA – determines the sequence of
amino acids to construct the polypeptide.
•If mRNA codon is
AUG, tRNA
anticodon would be
UAC
Ribosomal RNA
(rRNA)
• rRNA is made in
the nucleolus (a
cell structure
found inside the
nucleus)
• Ribosome made
of a large subunit
and small subunit
that join just prior
to protein
synthesis
• Ribosome has a binding site for mRNA and
binding sites for two tRNA molecules at a time.
• Several ribosomes may attach and translate the
same mRNA, therefore the name polyribosome
(letter c below).
Three Steps of Translation
1) Chain initiation
2) Chain elongation
3) Chain termination.
• Enzymes are required for each step, and
the first two steps require energy.
Animation
Animation
http://www.youtube.com/watch?v=D5vH4Q_tAkY
From video shown http://www.youtube.com/watch?v=41_Ne5mS2ls
in class:
Chain Initiation
• Small ribosomal
subunit attaches to
the mRNA near the
start codon.
• The anticodon of
tRNA, called the
initiator RNA, pairs
with the start codon
at the P site on
ribosome.
• Large ribosomal
subunit joins.
Chain Elongation
• The initiator tRNA passes its amino acid
to a tRNA-amino acid complex that has
come to the second binding site, the A
site.
• The ribosome moves forward and the
tRNA at the second binding site is now
at the first site, a sequence called
translocation.
• The previous tRNA leaves the ribosome
at the E site of the ribosome
Chain Termination
• A stop-codon is reached.
• A release factor (an enzyme) breaks the
polypeptide from the last tRNA
• The ribosome falls away from the mRNA
molecule and separates into its two
subunits
• A newly synthesized polypeptide may
function alone or become part of a
protein.
Review of Gene Expression
• DNA in the nucleus contains a triplet
code; each group of three bases stands for
one amino acid.
• During transcription, an mRNA copy of
the DNA template is made.
• The mRNA is processed before leaving
the nucleus.
• The mRNA joins with a ribosome, where
tRNA carries the amino acids into
position during translation.
Control of Gene Expression in Prokaryotes
The lac operon
•Regulator gene codes for active repressor,
which automatically attaches to the operator.
•RNA polymerase cannot attach to promoter,
and transcription does not occur
•When lactose attaches to repressor, it
becomes inactive and cannot attach to the
operator. Now RNA polymerase can attach
to the promoter, transcription occurs, and the
genes are expressed
Control of Gene Expression in Eukaryotes
• In eukaryotes, cells differ in which genes are
being expressed – based on cell function – ex.
nerve vs. muscle.
• One way that eukaryotes can control gene
expression is through proteins called
transcription factors
• Two types
• Those that guide and stabilize the binding
of RNA polymerase to a promotor
• Those that control the rate of transcription
(by controlling how DNA is folded or
preventing activators from binding)
• During development, cells become
specialized. This differentiation is
controlled by a set of genes called
Homeobox (Hox) genes.
• They code for transcription factors and
are active in specific parts of the DNA
corresponding to specific parts of the
body that is developing. They control
what body part will develop in a specific
location.
RNA interference:
• Some viruses have double-stranded
RNA
• An enzyme called dicer can cut this
RNA into small segments
• When they attach to protein complexes
in the cell, one of the strands breaks
down.
• The remaining section attaches to
molecules of mRNA, causing them to
break and preventing translation
Gene Mutations
• Definition - a change in the sequence of
bases within a gene
• Causes –
• Mutations can be spontaneous or caused by
environmental influences called mutagens.
• Cancer causing mutagens are called
carcinogens
• Mutagens include radiation (X-rays, UV
radiation), and organic chemicals (in
cigarette smoke and pesticides).
•Types –
•Frameshift mutations –
•one or more bases are inserted or
deleted from a sequence of DNA
•can result in nonfunctional proteins
•can result in no protein at all – stop
codon where there shouldn’t be one
•Point mutations –
•One base is substituted for another
•May result in change of amino acid
sequence
•May not affect protein at all
• Types of point mutations:
• Silent mutation - the change in the
codon results in the same amino acid
Ex: UAU  UAC both code for
tyrosine
• Nonsense mutation - a codon is
changed to a stop codon; resulting
protein may be too short to function
Ex: UAC  UAG (a stop codon)
• Missense mutation - involves the
substitution of a different amino acid, the
result may be a protein that cannot reach its
final shape
Ex: Hbs which causes sickle-cell disease
In general, mutations can have any of
the following effects:
•No change in proteins made or
appearance
•Wrong protein is made
•No protein in made
•New appearance may result
Repair of Mutations
•DNA polymerase proofreads the new strand
against the old strand and detects
mismatched pairs, reducing mistakes to one
in a billion nucleotide pairs replicated.
•If errors occur in sex cells – mutation may
be passed onto offspring
•If errors occur in body cells - cancer may
result
Transposons: Jumping Genes
• Transposons are specific DNA sequences
that move from place to place within and
between chromosomes.
• These “jumping genes” can cause a
mutation to occur by altering gene
expression.
• It is likely all organisms, including
humans, have transposons.