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Chapter 16-18
mini-lecture
Molecular Genetics
Ch 16: Molecular Basis of Inheritance
• 1953, James Watson and Francis Crick
proposed double-helix structure of DNA
Figure 16.1
History of DNA as the genetic material
• Griffith’s Transformation Experiment using
Streptococcus pneumoniae
– A bacterium that causes pneumonia in mammals
EXPERIMENTBacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they
have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule
and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:
Living S
(control) cells
Living R
(control) cells
Heat-killed
(control) S cells
Mixture of heat-killed S cells
and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample.
Figure 16.2
CONCLUSIONGriffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an
unknown, heritable substance from the dead S cells.
Bacteria Phase Experiments
• Hershey and Chase: DNA as the genetic
material!
Phage
head
Tail
Tail fiber
Figure 16.3
Bacterial
cell
100 nm
DNA
Hershey and Chase
EXPERIMENT In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur
and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.
1 Mixed radioactively
labeled phages with
bacteria. The phages
infected the bacterial cells.
Phage
2 Agitated in a blender to
separate phages outside
the bacteria from the
bacterial cells.
Radioactive
protein
3 Centrifuged the mixture
so that bacteria formed
a pellet at the bottom of
the test tube.
Empty
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1: Phages were
grown with radioactive
sulfur (35S), which was
incorporated into phage
protein (pink).
Batch 2: Phages were
grown with radioactive
phosphorus (32P), which
was incorporated into
phage DNA (blue).
4 Measured the
radioactivity in
the pellet and
the liquid
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Centrifuge
Radioactivity
(phage DNA)
Pellet
in pellet
RESULTS Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells.
When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.
Figure 16.4
CONCLUSION
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
1947
• Maurice Wilkins and Rosalind Franklin
– Were using a technique called X-ray crystallography
to study molecular structure
• Rosalind Franklin
– Produced a picture of the DNA molecule using this
technique
Figure 16.6 a, b
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
Photograph of DNA
Watson and Crick
DNA as double-helix
Base pairing rules and H-bonds
G
H
N
N
N
C
A
N
T
T
Sugar
A
O
H
H
N
N
N
1 nm
3.4 nm
G
A
Adenine (A)
T
H
G
C
T
O
N
A
T
H
N
A
A
T
G
A
N
T
A
Figure 16.7a, c
O
Su
Thymine (T
C
G
C
C
Sugar
0.34 nm
T
(a) Key features of DNA structure
(c) Space-filling model
Figure 16.8
N
H
N
N
N
N
H
Guanine (G)
H
O
S
Cytosine
DNA Replication and Repair
Semi-conservation!
T
A
T
A
T
A
C
G
C
G
C
T
A
T
A
T
A
A
T
A
T
A
T
G
C
G
C
G
C
G
A
T
A
T
A
T
C
G
C
G
C
G
T
A
T
A
T
A
T
A
T
A
T
C
G
C
G
C
A
G
(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
Figure 16.9 a–d
(b) The first step in replication is
separation of the two DNA
strands.
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
Meselson and Stahl
EXPERIMENT Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations
on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria
incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with
only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be
lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different
densities by centrifuging DNA extracted from the bacteria.
1
Bacteria
cultured in
medium
containing
15N
2
Bacteria
transferred to
medium
containing
14N
RESULTS
3
DNA sample
centrifuged
after 20 min
(after first
replication)
4 DNA sample
centrifuged
after 40 min
(after second
replication)
Less
dense
More
dense
The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask
Figure 16.11 in step 2, one sample taken after 20 minutes and one after 40 minutes.
DNA Replication in Detail
Leading vs. Lagging Strands
1 DNA pol Ill elongates
DNA strands only in the
5
3 direction. 3
5
Parental DNA
5
3
Okazaki
fragments
2
1
3
5
DNA pol III
2 One new strand, the leading strand,
can elongate continuously 5
3
as the replication fork progresses.
3 The other new strand, the
lagging strand must grow in an overall
3
5 direction by addition of short
segments, Okazaki fragments, that grow
5
3 (numbered here in the order
they were made).
Template
strand
3
Leading strand
Lagging strand
Template
strand
Figure 16.14
2
1
DNA ligase
Overall direction of replication
4 DNA ligase joins Okazaki
fragments by forming a bond between
their free ends. This results in a
continuous strand.
1
Primase joins RNA nucleotides
into a primer.
3
5
5
3
Template
strand
RNA primer
3
5
3
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
2
5
3
1
After reaching the next
RNA primer (not shown),
DNA pol III falls off.
Okazaki
fragment
3
3
5
1
5
4
After the second fragment is
primed. DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off.
5
3
5
3
2
5
1
DNA pol 1 replaces the
RNA with DNA, adding to
the 3 end of fragment 2.
5
3
6
5
1
DNA ligase forms a bond
between the newest DNA
and the adjacent DNA of
fragment 1.
5
3
Figure 16.15
3
2
7
The lagging strand
in this region is now
complete.
3
2
1
Overall direction of replication
5
Other Proteins That Assist DNA Replication
• Helicase, topoisomerase, single-strand binding
protein
– Are all proteins that assist DNA replication
Table 16.1
Proofreading and Repair
• DNA Polymerase has
proofreading ability
• Repair enzyme
corrects base pairing
– Nucleotide excision
repair
Figure 16.17
1 A thymine dimer
distorts the DNA molecule.
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
Replicating the Ends of DNA Molecules
• The ends of eukaryotic chromosomal DNA
– Get shorter with each round of replication
5
End of parental
DNA strands
Leading strand
Lagging strand
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Figure 16.18
Shorter and shorter
daughter molecules
Figure 16.19
1 µm
Chapter 17
Figure 17.6
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a jellyfish
gene
Figure 17.7-4
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
2 Elongation
Rewound
DNA
5
3
3
5
3
5
RNA
transcript
3 Termination
3
5
5
3
5
Completed RNA transcript
3
Direction of transcription (“downstream”)
Figure 17.8
1 A eukaryotic promoter
Promoter
Nontemplate strand
DNA
5
3
3
5
T A T A A AA
A T AT T T T
TATA box
Transcription
factors
Start point
Template strand
2 Several transcription
factors bind to DNA
5
3
3
5
3 Transcription initiation
complex forms
RNA polymerase II
Transcription factors
5
3
5
3
RNA transcript
Transcription initiation complex
3
5
Figure 17.9
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
A
3
T
C
C
A A
5
3 end
C A
U
C
C A
T
A
G
G T
5
5
C
3
T
Direction of transcription
Template
strand of DNA
Newly made
RNA
Figure 17.11
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
130
31104
numbers
Intron
Exon 3
Poly-A tail
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
Coding
segment
3 UTR
Figure 17.12-3
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
Figure 17.13
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Figure 17.14
Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
tRNA
C
G
Anticodon
U G G U U U G G C
5
Codons
mRNA
3
Figure 17.17
Growing
polypeptide
tRNA
molecules
E P
Exit tunnel
Large
subunit
A
Small
subunit
5
mRNA
3
(a) Computer model of functioning ribosome
Growing polypeptide
P site (Peptidyl-tRNA
binding site)
Exit tunnel
Next amino
acid to be
added to
polypeptide
chain
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
Amino end
P
A
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
E
tRNA
mRNA
5
3
Codons
(c) Schematic model with mRNA and tRNA
Figure 17.20-3
Release
factor
Free
polypeptide
5
3
3
5
5
Stop codon
(UAG, UAA, or UGA)
2
GTP
2 GDP  2 P i
3
Figure 17.21
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
(a)
End of
mRNA
(3 end)
Ribosomes
mRNA
(b)
0.1 m
Figure 17.22
1 Ribosome
5
4
mRNA
Signal
peptide
3
SRP
2
ER
LUMEN
SRP
receptor
protein
Translocation
complex
Signal
peptide
removed
ER
membrane
Protein
6
CYTOSOL
Figure 17.25
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase
Direction of
transcription
0.25 m
DNA
Polyribosome
Polypeptide
(amino end)
Ribosome
mRNA (5 end)
Figure 17.26
DNA
TRANSCRIPTION
3
5
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
AminoacyltRNA synthetase
Intron
NUCLEUS
Amino
acid
AMINO ACID
ACTIVATION
tRNA
CYTOPLASM
mRNA
Growing
polypeptide
3
A
Aminoacyl
(charged)
tRNA
P
E
Ribosomal
subunits
TRANSLATION
E
A
Anticodon
Codon
Ribosome
Figure 18.3
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpR
trpD
trpC
trpB
trpA
C
B
A
Operator
Regulatory
gene
3
RNA
polymerase
Start codon
Stop codon
mRNA 5
mRNA
5
E
Protein
Inactive
repressor
D
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
Figure 18.3b-2
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
Figure 18.4
Regulatory
gene
DNA
Promoter
Operator
lacI
lacZ
No
RNA
made
3
mRNA
RNA
polymerase
5
Active
repressor
Protein
(a) Lactose absent, repressor active, operon off
lac operon
DNA
lacI
lacZ
lacY
lacA
RNA polymerase
3
mRNA
5
mRNA 5
-Galactosidase
Protein
Allolactose
(inducer)
Inactive
repressor
(b) Lactose present, repressor inactive, operon on
Permease
Transacetylase
Figure 18.4a
Regulatory
gene
DNA
Promoter
Operator
lacI
lacZ
No
RNA
made
3
mRNA
5
Protein
RNA
polymerase
Active
repressor
(a) Lactose absent, repressor active, operon off
Figure 18.5
Promoter
DNA
lacI
lacZ
CAP-binding site
cAMP
Operator
RNA
polymerase
Active binds and
transcribes
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
Promoter
DNA
lacI
CAP-binding site
lacZ
Operator
RNA
polymerase less
likely to bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized
Figure 18.6
Signal
NUCLEUS
Chromatin
DNA
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
Gene available
for transcription
Gene
Transcription
RNA
Exon
Primary transcript
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Degradation
of protein
Active protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)
Figure 18.7
Histone
tails
Amino acids
available
for chemical
modification
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Acetylated histones
Unacetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription
Figure 18.8-3
Enhancer
(distal control
elements)
Proximal
control
elements
Transcription
start site
Exon
DNA
Upstream
Intron
Exon
Intron
Downstream
Poly-A
signal
Intron Exon
Exon
Cleaved
3 end of
primary
RNA processing
transcript
Promoter
Transcription
Exon
Primary RNA
transcript
5
(pre-mRNA)
Poly-A
signal Transcription
sequence termination
region
Intron Exon
Intron RNA
Coding segment
mRNA
G
P
AAA AAA
P P
5 Cap
5 UTR
Start
Stop
codon codon
3 UTR Poly-A
tail
3
Figure 18.10-3
Promoter
Activators
DNA
Enhancer
Distal control
element
Gene
TATA box
General
transcription
factors
DNAbending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Figure 18.11
Enhancer
Control
elements
Promoter
Albumin gene
Crystallin
gene
LENS CELL
NUCLEUS
LIVER CELL
NUCLEUS
Available
activators
Available
activators
Albumin gene
not expressed
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Crystallin gene
expressed
(b) Lens cell
Figure 18.13
Exons
DNA
1
3
2
4
5
Troponin T gene
Primary
RNA
transcript
3
2
1
5
4
RNA splicing
mRNA
1
2
3
5
or
1
2
4
5
Figure 18.14
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
Proteasome
and ubiquitin
to be recycled
Protein entering
a proteasome
Protein
fragments
(peptides)
Figure 18.15
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
miRNAprotein
complex
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
Figure 18.23
Proto-oncogene
DNA
Translocation or
transposition: gene
moved to new locus,
under new controls
Gene amplification:
multiple copies of
the gene
New
promoter
Normal growthstimulating
protein in excess
Point mutation:
within a control
within
element
the gene
Oncogene
Normal growth-stimulating
protein in excess
Normal growthstimulating
protein in
excess
Oncogene
Hyperactive or
degradationresistant
protein
Figure 18.24b
2 Protein kinases
3 Active
form
of p53
UV
light
1 DNA damage
in genome
DNA
Protein that
inhibits
the cell cycle
(b) Cell cycle–inhibiting pathway
MUTATION
Defective or missing
transcription factor,
such as
p53, cannot
activate
transcription.