Download Molecular Genetics

Molecular Genetics
Part One
AP Biology
1908 | 1933
Chromosomes related to phenotype
 T.H. Morgan

working with Drosophila
 fruit flies

associated phenotype with
specific chromosome
 white-eyed male had specific
X chromosome
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1908 | 1933
Genes are on chromosomes
 Morgan’s conclusions
genes are on chromosomes
 but is it the protein or the
DNA of the chromosomes
that are the genes?

 initially proteins were thought
to be genetic material…
Why?
What’s so impressive
about proteins?!
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The “Transforming Principle”
 Frederick Griffith

Streptococcus pneumonia bacteria
 was working to find cure for pneumonia
harmless live bacteria (“rough”)
mixed with heat-killed pathogenic
bacteria (“smooth”) causes fatal
disease in mice
 a substance passed from dead
bacteria to live bacteria to change
their phenotype


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“Transforming Principle”
1928
The “Transforming Principle” mix heat-killed
live pathogenic
strain of bacteria
A.
mice die
live non-pathogenic heat-killed
strain of bacteria
pathogenic bacteria
B.
C.
mice live
mice live
pathogenic &
non-pathogenic
bacteria
D.
mice die
Transformation = change in phenotype
something in heat-killed bacteria could still transmit
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disease-causing properties
1944
DNA is the “Transforming Principle”
 Avery, McCarty & MacLeod

purified both DNA & proteins separately from
Streptococcus pneumonia bacteria
 which will transform non-pathogenic bacteria?

injected protein into bacteria
 no effect

injected DNA into bacteria
 transformed harmless bacteria into
virulent bacteria
mice die
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What’s the
conclusion?
1944 | ??!!
Avery, McCarty & MacLeod
 Conclusion

first experimental evidence that DNA was the
genetic material
Oswald Avery
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Maclyn McCarty
Colin MacLeod
1952 | 1969
Confirmation of DNA
 Hershey & Chase
classic “blender” experiment
 worked with bacteriophage

 viruses that infect bacteria

Why use
Sulfur
vs.
Phosphorus?

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grew phage viruses in 2 media,
radioactively labeled with either

35S
in their proteins
 32P in their DNA
infected bacteria with
labeled phages
Hershey
Protein coat labeled
with 35S
Hershey
& Chase
DNA labeled with 32P
T2 bacteriophages
are labeled with
radioactive isotopes
S vs. P
bacteriophages infect
bacterial cells
bacterial cells are agitated
to remove viral protein coats
Which
radioactive
marker is found
inside the cell?
Which molecule
carries viral
genetic
info?
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35S
radioactivity
found in the medium
32P
radioactivity found
in the bacterial cells
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Blender experiment
 Radioactive phage & bacteria in blender

35S
phage
 radioactive proteins stayed in supernatant
 therefore viral protein did NOT enter bacteria
 32
P phage
 radioactive DNA stayed in pellet
 therefore viral DNA did enter bacteria

Confirmed DNA is “transforming factor”
Taaa-Daaa!
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1952 | 1969
Hershey
Hershey & Chase
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Martha Chase
Alfred Hershey
1947
Chargaff
 DNA composition: “Chargaff’s rules”
varies from species to species
 all 4 bases not in equal quantity
 bases present in characteristic ratio

 humans:
A = 30.9%
T = 29.4%
G = 19.9%
C = 19.8%
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That’s interesting!
What do you notice?
Rules
A = T
C = G
1953 | 1962
Structure of DNA
 Watson & Crick

developed double helix model of DNA
 other leading scientists working on question:
 Rosalind Franklin
 Maurice Wilkins
 Linus Pauling
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Franklin
Wilkins
Pauling
1953 article in Nature
Watson and Crick
Watson
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Crick
Rosalind Franklin (1920-1958)
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A chemist by training, Franklin had made original and essential contributions to the
understanding of the structure of graphite and other carbon compounds even before her
appointment to King's College. Unfortunately, her reputation did not precede her. James
Watson's unflattering portrayal of Franklin in his account of the discovery of DNA's
structure, entitled "The Double Helix," depicts Franklin as an underling of Maurice Wilkins,
when in fact Wilkins and Franklin were peers in the Randall laboratory. And it was Franklin
alone whom Randall had given the task of elucidating DNA's structure. The technique with
which Rosalind Franklin set out to do this is called X-ray crystallography. With this
technique, the locations of atoms in any crystal can be precisely mapped by looking at the
image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning
how to use this technique to study biological molecules. Rosalind Franklin applied her
chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she
discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on
the outside of the molecule. She also elucidated the basic helical structure of the molecule.
After Randall presented Franklin's data and her unpublished conclusions at a routine
seminar, her work was provided - without Randall's knowledge - to her competitors at
Cambridge University, Watson and Crick. The scientists used her data and that of other
scientists to build their ultimately correct and detailed description of DNA's structure in
1953. Franklin was not bitter, but pleased, and set out to publish a corroborating report of
the Watson-Crick model. Her career was eventually cut short by illness. It is a tremendous
shame that Franklin did not receive due credit for her essential role in this discovery, either
during her lifetime or after her untimely death at age 37 due to cancer.
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But how is DNA copied?
 Replication of DNA

base pairing suggests
that it will allow each
side to serve as a
template for a new
strand
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
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material.
”
— Watson & Crick
Problem:
How does DNA replicate?
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Replication: Making DNA from existing DNA
3 alternative
models of DNA
replication
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Meselson & Stahl
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Meselson & Stahl
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Scientific History
 March to understanding that DNA is the genetic material

T.H. Morgan (1908)
 genes are on chromosomes

Frederick Griffith (1928)
 a transforming factor can change phenotype

Avery, McCarty & MacLeod (1944)
 transforming factor is DNA

Erwin Chargaff (1947)
 Chargaff rules: A = T, C = G

Hershey & Chase (1952)
 confirmation that DNA is genetic material

Watson & Crick (1953)
 determined double helix structure of DNA

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Meselson & Stahl (1958)
 semi-conservative replication
The “Central Dogma”
 Flow of genetic information in a cell
transcription
DNA
replication
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translation
RNA
protein
Science …. Fun
Party Time!
Any Questions??
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DNA REPLICATION (Animation 2)
 DNA Structure
Sugar
 Phosphodiester
bond
 Phosphate
 Bases

 Purines
 Pyramidines

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The strands are antiparallel
1. Helicase unwinds DNA at origins of replication and
creates replication forks
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 Prokaryotic Cells

Single RF
 Eukaryotic Cells

100s of RF
 Replication will proceed in both
directions
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 2. Single stranded binding proteins
attach to each DNA strand to stabilize
them
 3. Topoisomerase binds to the twisted
ends of DNA to prevent supercoiling
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4. Primase adds RNA primer at the origin of replication
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 5. DNA polymerase III attaches to the primer
 6. On the leading strand, DNA polymerase III,
running along the 3’ 5’ DNA strand, adds
nucleotides to the free 3’ end of nucleotides



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Builds 5’  3’
DNA polymerase moves towards the
replication fork
Nucleotide triphosphates power DNA
replication
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Replication on leading strand
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 7. Along the lagging strand
The one which DNA pol III runs away
from the replication fork) DNA
fragments are synthesized in short
okazaki fragments
 Primase RNA primer  DNA pol III 
Okazaki fragments

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 8. DNA polymerase I removes primer
and compliments DNA nucleotides
 9. DNA ligase seals the phosphodiester
bonds between the newly added
nucleotides
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Okazaki Fragments:
Short segments of DNA
that grow 5’3’ that are
added onto the Lagging
Strand
DNA Ligase: seals
together fragments
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Major Steps of Replication:
1. Helicase: unwinds DNA at origins of replication
2. Initiation proteins separate 2 strands  forms
replication bubble
3. Primase: puts down RNA primer to start replication
4. DNA polymerase III: adds complimentary bases to
leading strand (new DNA is made 5’  3’)
5. Lagging strand grows in 3’5’ direction by the
addition of Okazaki fragments
6. DNA polymerase I: replaces RNA primers with DNA
7. DNA ligase: seals fragments together
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Leading strand vs. Lagging strand
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The ends of DNA molecules pose a
special problem
 Because DNA polymerases can only add
nucleotides to the 3' end of a preexisting
polynucleotide, repeated replication of linear
DNA, such as that possessed by all eukaryotes,
would result in successively shorter molecules,
potentially deleting genes
 • Prokaryotes don't have this problem because
they possess circular DNA.
 • Eukaryotic DNA is flanked by telomeres,
repeats of short noncoding nucleotide
sequences.
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Molecular Genetics
Part Deux
AP Biology
1941 | 1958
Beadle & Tatum
one gene : one enzyme hypothesis
George Beadle
Edward Tatum
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"for their discovery that genes act by
regulating definite chemical events"
Beadle & Tatum
X rays or ultraviolet light
Wild-type
Neurospora
create mutations
asexual
spores
Minimal
medium
spores
Growth on
complete
medium
positive control
Select one of
the spores
Test on minimal
medium to confirm
presence of mutation
negative control
Grow on
complete medium
Minimal media supplemented only with…
experimentals
Choline
Pyridoxine
Riboflavin
Minimal
Nucleic
Arginine
control
amino acid p-Amino
Niacin
Inositol acid Folic
supplements
acid
Thiamine
benzoic acid
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 Newer idea: one gene-one
polypeptide hypothesis
Most accurate: one gene-one
RNA molecule (which can be
translated into a polypeptide)
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a
a
From gene to protein
nucleus
cytoplasm
transcription
DNA
a
a
translation
mRNA
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
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Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
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Flow of Genetic
Information in
Prokaryotes vs.
Eukaryotes
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RNA
 ribose sugar
 N-bases
uracil instead of thymine
U : A
C : G

 single stranded
 lots of RNAs

DNA
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mRNA, tRNA, rRNA, snRNA…
transcription
RNA
 rRNA – Ribosomal RNA – comprised of
protein and rRNA
 Small Nucleotide RNA (snRNA) – comprise
sliceosomes
 MicroRNA – involved in process called RNA
interference
 Ribozymes – RNA can fold into an enzymelike molevule to catalyze its own replication
 tRNA – transport individual amino acids to
the ribosome
 mRNA – intermediate genetic material
between DNA + protein synthesis
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Transcription Factors
 Initiation complex

transcription factors bind to promoter
region
 suite of proteins which bind to DNA

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trigger the binding of RNA polymerase to
DNA
1. Initiation
Bacteria: RNA
polymerase binds
directly to promoter
in DNA
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1. Initiation
Eukaryotes:
TATA box = DNA
sequence (TATAAAA)
upstream from
promoter
Transcription
factors must
recognize TATA box
before RNA
polymerase can
bind to DNA
promoter
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2. Elongation
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• RNA polymerase
adds RNA nucleotides
to the 3’ end of the
growing chain (A-U, GC)
• ATP powers RNA
ploymerase to run
along the 3’  5’ DNA
strand
2. Elongation
As RNA polymerase
moves, it untwists DNA,
then rewinds it after
mRNA is made
mRNA is a 5’-3’
prime strand
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3. Termination
RNA polymerase
transcribes a terminator
sequence in DNA, then
mRNA and polymerase
detach.
It is now called pre-mRNA
for eukaryotes.
Prokaryotes = mRNA ready
for use
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Flow of Genetic
Information in
Prokaryotes vs.
Eukaryotes
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4a) mRNA Processing
 After transcription begins, a methylated
GTC cap - 5’ cap – “guanine cap” is added to
the 5’ end of the mRNA strand.
 3’ poly-A tail (50-520 A’s) are added by
poly A polymerase to the 3’ tail
 Proteins are also attached to the tail
 Help export from nucleus, protect from
enzyme degradation, attach to ribosomes
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4b) mRNA Splicing
 Pre-mRNA has introns (noncoding sequences)
and exons (codes for amino acids)
 Splicing = introns cut out, exons joined together
 Animation snRNP
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Draw on Board
1. Small-nucleotide riboproteins (snRNPs)
attach to both the 5’ and 3’ ends of an
intron
2. The snRNPs join to create a spliceosome
3. The spliceosome cleaves the intron and
ligates the exon segments together
4.
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Why have introns?
 Some regulate gene
activity
 Alternative RNA Splicing:
produce different
combinations of exons
One gene can make
more than one
polypeptide!
 20,000 genes 
100,000
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polypeptides

TRANSLATION
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Components of Translation
1. mRNA = message
2. tRNA = interpreter
3. Ribosome = site of
translation
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tRNA
 Transcribed in nucleus
 Specific to each amino acid
 Transfer AA to ribosomes
 Anticodon: pairs with
complementary mRNA
codon
 Base-pairing rules between
3rd base of codon &
anticodon are not as strict.
This is called wobble.
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tRNA
 Aminoacyl-tRNAsynthetase: enzyme that
binds tRNA to specific
amino acid
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Ribosomes
 Ribosome = rRNA +
proteins
 made in nucleolus
 2 subunits
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Ribosomes
Active sites:
 A site: holds AA to be added
 P site: holds growing
polypeptide chain
 E site: exit site for tRNA
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Translation:
1. Initiation
• Small subunit binds to start codon (AUG) on mRNA
• tRNA carrying Met attaches to P site
• Large subunit attaches
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2. Elongation
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2. Elongation
Codon recognition:
tRNA anticodon
matches codon in A
site
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2. Elongation
Peptide bond
formation: AA in A
site forms bond with
peptide in P site
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2. Elongation
Translocation: tRNA
in A site moves to
P site; tRNA in P
site moves to E site
(then exits)
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2. Elongation
Repeat over
and over
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3.Termination
 Stop codon reached and translation stops
 Release factor binds to stop codon; polypeptide
is released
 Ribosomal subunits dissociate
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The code
 Code for ALL life!
strongest support for
a common origin for
all life

 Code is redundant
several codons for
each amino acid
3rd base “wobble”


Why is the
wobble good?
 Start codon


AUG
methionine
 Stop codons
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
UGA, UAA, UAG
How are the codons matched to
amino acids?
DNA
TACGCACATTTACGTACGCGG
3
5
codon
mRNA
5
AUGCGUGUAAAUGCAUGCGCC
3
5
tRNA
UAC GCA CAU
amino
acid
Met Arg Val
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anti-codon
3
Polyribosomes
 A single mRNA
can be translated
by several
ribosomes at the
same time
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Protein Folding (Review)
 During synthesis, polypeptide chain coils and
folds spontaneously
 Chaperonin: protein that helps polypeptide fold
correctly
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Types of Ribosomes
 Free ribosomes: synthesize proteins that stay in
cytosol and function there
 Bound ribosomes (to ER): make proteins of
endomembrane system (nuclear envelope, ER,
Golgi, lysosomes, vacuoles, plasma
membrane) & proteins for secretion
 Uses signal peptide to target location
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Cellular “Zip Codes”
 Signal peptide: 20 AA at leading end of
polypeptide determines destination
 Signal-recognition particle (SRP): brings
ribosome to ER
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The Central Dogma
Mutations happen here
Effects play out here
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Mutations = changes in the genetic
material of a cell
 Large scale mutations: chromosomal; always cause

disorders or death
 nondisjunction, translocation, inversions,
duplications, large deletions
Point mutations: alter 1 base pair of a gene
1. Base-pair substitutions – replace 1 with another
 Missense: different amino acid
 Nonsense: stop codon, not amino acid
2. Frameshift – mRNA read incorrectly; nonfunctional
proteins
 Caused by insertions or deletions
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Substitution = Missense
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Substitution = Nonsense
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Substitution = Silent (no effect)
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Insertion = Frameshift Mutation
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Deletion = Extensive missense,
premature termination
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DNA Repair
 Excision (Dark) Repair



A)”Profreading Enzyme” detects mutation
B) nuclease cuts loops 10 bps upstream (5’
end) and 10 bps downstream to remove
segment
C)DNA polymerase replaces nucleotides, DNA
ligase bonds
 Light Repair

Replaces Thymine Dimer
 2 adjacent thymines covalently bond due to light (UV
Radiation)
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