Chapter 14:
DNA: The Genetic Material
I. Historical Experiments
II. The structure of DNA
III. DNA Replication
I. Historical Experiments
•
The structure of DNA was worked out only recently: 1950’s
•
The “discovery” of DNA structure & function occurred in stages:
Late 1800’s:
1928:
1930’s:
1944-1952
1952
heritable info existed in discrete units
= genes; knew of “nuclein” (aka DNA)
Hereditary information can pass from dead
cells to live cells (Griffith)
Hereditary information is thought to be stored
in the nucleus (Hammerling)
DNA is the molecule of heredity; not proteins
(Avery/Hershey-Chase)
Confirmation that hereditary information is
contained in the nucleus; totipotency
(Briggs and King)
1
Griffith Experiments – 1920’s
Hereditary information can pass from dead cells
to living cells, transforming them
Research by Frederick Griffith
Trying to find pneumonia vaccine
Vaccines are often strains
(types) of bacteria or viruses
which are:
- heat killed
- weakened
- harmless
• Injected into the body
• Promote immunity against
disease causing strains
Griffith’s work: Link between DNA and genes
2
What was that substance?
Years later (1944):
• Oswald Avery
–
–
–
•
Used enzymes to Destroy, DNA, RNA or Protein in
bacterial extracts.
If he destroyed everything BUT the DNA he got
transformation
If he destroyed the DNA, NO transformation
Introduced concept that genes in DNA code for
characteristics
Hershey & Chase – 1952
Bacteriophage: a virus that attacks bacteria
Protein
Head
DNA
Tail
300,000!
Tail fiber
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
3
Hershey & Chase – 1952
Bacteriophage reproductive cycle
Phage
attaches
to bacterial
cell.
Phage
injects
DNA.
Phage DNA directs cell to
1. make more phage DNA
2. make protein parts.
New phages assemble.
Cell lyses
releasing
new
phages.
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Hershey & Chase – 1952
T2 bacteriophages
labeled with
radioactive isotopes
Protein coat
labeled
with 35S
DNA labeled
with 32P
Bacteriophages infect
bacterial cells.
Bacterial cells agitated
to remove protein coats.
35S
radioactivity
found in the medium
32P
radioactivity found
in the bacterial cells
DNA IS THE GENETIC MATERIAL!
4
II. The structure of DNA
•
•
•
All DNA composed of monomeric subunits
These are nucleotides
Each nucleotide has 3 parts:
(1) Phosphate group
(2) Deoxyribose sugar
(3) one of four possible nitrogen
containing bases:
Thymine
Cytosine
= four different
Adenine
nucleotides
Guanine
Nucleotide Structure
PO4
Nitrogen
Base
5’ CH2
O
1’
4’
3’
2’
OH
Carbons are numbered
5
Phosphodiester bond (dehydration synthesis)
5!
Nucleotide
Polymer
PO4
5’ Carbon
CH2
O
C
3’ –OH group
Phosphate group
Base
–O
O
P O
O
CH2
Base
O
3’ –OH group
OH
3!
DNA is made of
repeating
subunits
Chargaff’s rules
1. Proportion of A equals that of T, proportion of G
equals that of C
2. Therefore, equal proportion of purines and
pyrimidines
6
Rosalind Franklin and Maurice Wilkins: X-Ray Diffraction
1. DNA was helical
2. Franklin deserves credit for determining the dimensions
of DNA
3. Diameter was 2 nm; complete helical turn every 3.4 nm
4. Distance between base pairs 0.34 nm
Watson & Crick: Structure of DNA (1953)
• Each strand has a “free”
phosphate end and sugar
end (5’ and 3’)
• Strands run in opposite
directions ! antiparallel
7
Watson & Crick: Structure of DNA (1953)
• H-bonds between
complimentary bases hold
strands together
• Complementary base pairs:
Adenine pairs with Thymine
Cytosine pairs with Guanine
• Purine w/ Pyrmidine; only
combination that gives
correct width
•A – T: 2 H-bonds
•G – C: 3 H-bonds
Watson & Crick:
Structure of DNA (1953)
2 nm
5!
3!
AT
TA
Minor groove
Major groove
GC
C G
T A
G C
T A
G C
AT
TA
3.4 nm
0.34 nm
G C
C G
T A
G C
8
Summary
DNA Structure
1. Two nucleotide chains
sugar-phosphate backbone
2. Base Complementarity
A-T
and
G-C
3. Double Helix
4. Antiparallel
3’ – 5’ ends opposite
5. Each strand is a
reverse complement of
the other strand
III. DNA replication
•
Cells must synthesize copies of their DNA prior to
division
Basic idea:
1) Enzymes pull apart parental
double helix
2) Another enzyme joins free
nucleotides to complement
each strand
3) When finished, have 2
double helices
–
each with 1 old and 1 new
strand
9
DNA Replication
1. Stages: Initiation ! Elongation ! Termination
a. Initiation: always occurs at the same site
b. Elongation: majority of replication
c. Termination: synthesis ends
2. Key enzymes
1. Helicase – breaks H-bonds, opens helix
2. DNA Primase – makes RNA primer strand
3. DNA Polymerase – adds nucleotides
4. Ligase – seals up breaks in sugar/P backbone
3. DNA Synthesis is both Continuous &
Discontinuous
Stages: Initiation ! Elongation ! Termination
Parental DNA
Replication fork
New
strands
1. INITIATION
Replication origin
2. ELONGATION
Template strands
3. TERMINATION
Two daughter
DNA duplexes
10
DNA replication is “semi-conservative”
Meselson—Stahl
Experiments
Used a heavy
isotope of N to track
parental and
daughter strands
DNA replication is “semi-conservative”
Meselson—Stahl
Experiments
Used a heavy
isotope of N to track
parental and
daughter strands
11
DNA replication is “semi-conservative”
DNA replication is “semi-conservative”
H/L
H/L & L/L
H/L & L/L
12
The Mechanism of DNA Replication
DNA polymerase III adds nucleotides to 3’ end Template
New
strand
strand
HO 3’
HO 3’
5’
5’
C G
C G
Template
P New
P
O
strand
O
strand
O
O
P
P
T
T
A
A
O P
O P
O
O
DNA
P
P
A T
A T
P Polymerase
O P
III
O
O
O
P
P
C G
C G
P
O P
O
O
O
P
P
A
A T
3’ OH
P PP
O
O
O
T
P
P
O PPP
A
A
3’ OH
O
O
OH
P
P
5’
5’
The Mechanism of DNA Replication
• DNA polymerase adds nucleotides ONLY to the 3’
end; requires a primer sequence to begin
• Occurs in an assembly called the replisome
– The following proteins are involved:
• DNA helicase – opens helix
• Single-strand binding proteins – keep strands
separate (stability)
• DNA primase – makes RNA primer strand
• DNA polymerase – adds nucleotides
• DNA ligase – seals up breaks in sugar/P backbone
• DNA gyrase – relieves torque
13
The Mechanism of DNA Replication
Helicase breaks Hbonds; may have
several replication
forks going at once
DNA Polymerase
DNA Ligase
BUT, DNA polymerase can only add free nucleotides to the
free sugar end of the DNA strand (5’ to 3’)
Leading Strand
Continuous
Synthesis
3’
DNA
polymerase III
Unwinding
5’
Okazaki fragment
3’
Lagging strand
Discontinuous
Synthesis
3’
5’
Will be joined later by Ligase
14
DNA polymerase can only add free nucleotides to an
existing chain ! needs a “primer” sequence
DNA Primase
Putting it all together
15
Eukaryotic
DNA
Replication
Parent strand
Daughter
strand
1. More DNA
1
2. Numerous
Replication
Forks
3. Names of the
enzymes may
differ from
prokaryotic
replication
Point of
separation
2
3
4
Do mistakes happen?
• Initially: 1/10,000 base pairs.
– Not bad for rate of 700
nucleotides added/minute
• After “proofreading”:
– 1 per billion base pairs
Why so few?
• Each new strand is
complimentary to the
parental strand
• H-bonding between bases is
extremely specific
A-T & C-G
• Exonuclease activity of DNA
polymerase III
16
What else might cause mistakes/changes to DNA?
• Environmental factors
– i.e., UV rays in sunlight
• Causes “rearrangement” of H-bonds between bases
• Usually corrected by repair enzymes
– Too much damage...
• Skin cell suicide = peeling sunburn
Or
• Melanoma: skin cancer
Chapter 15:
Genes and How they Work
I.
II.
III.
IV.
V.
VI.
Gene Expression
Genetic Code
Eukaryotic Transcription
Posttranscriptional Modification
Translation
Comparison of Gene Expression
in Prokaryotes and Eukaryotes
VII. Structure of a Eukaryotic Gene
VIII. Control of Gene Expression
17
I. Gene Expression
A. Most individual genes (in DNA) contain information to
synthesize proteins (chains of amino acids;
polypeptides)
Genes Specify Sequences of Amino Acids
Normal hemoglobin " chain (Sanger 1953)
Valine
Histidine
Leucine
Threonine
Proline
Valine
Histidine
Leucine
Threonine Proline
Glutamic
acid
Glutamic
acid
Valine
Glutamic
acid
Sickle cell anemia hemoglobin " chain (Ingram 1956)
GENE = Unit of Heredity
Sequence of nucleotides that
determines the amino acid
sequence of a protein
I. Gene Expression
B. Where are genes and proteins located?
•
•
DNA remains in nucleus (in chromatin)
Protein synthesis takes place in cytoplasm
Ribosomes: protein synthesizers
C. Information transferred with:
Ribonucleic Acid (RNA)
•
•
•
•
Single-stranded
Ribose sugar
has A, C, G nucleotides
Lacks thymine nucleotides:
– has uracil instead
18
What is the flow of gene-encoded information?
Central Dogma
The Central Dogma traces the flow of geneencoded information
I. Gene Expression
Three types of RNA are made by cells
1) mRNA: Messenger RNA
2) rRNA: Ribosomal RNA
3) tRNA: Transfer RNA
19
II. Genetic Code
A. How does RNA code for proteins?
RNA sequence --> Amino acid sequence
Nucleotide sequence is in genetic code
! In RNA (or DNA) there are 4 different bases
= 4 different nucleotides
II. Genetic Code- “Universal?”
20
II. Genetic Code
C. The language of RNA
Protein
= sentence of codon words
(codons code for amino acids)
Punctuation of sentence:
Start codon = AUG = Methionine
Stop codon = “period.”
All proteins end with 1 of 3 stop codons
64 possible codons - 3 stop codons = 61 AA codons
Most AA coded for by multiple codons ! redundancy
III. Eukaryotic Transcription
Steps in Transcription
(1) Initiation
• RNA polymerase binds at
promoter of gene on DNA
(2) Elongation
• Transcribes 1 direction on
template DNA strand
• Makes complimentary
RNA strand
(3) Termination
• Terminator sequence
• RNA polymerase detaches
21
DNA Promoter Sites are Start sites
Transcription factor
TATA box of DNA promoter
Eukaryotic DNA
TATAAA
1. A transcription factor
recognizes and binds to the
TATA box sequence which is
part of the core DNA promoter.
A promoter is a DNA sequence that enables a gene to be transcribed.
Promoters are a means to demarcate which regions of the DNA should be
used for messenger RNA creation - and, by extension, control which
proteins the cell manufactures.
2. Other transcription factors
are recruited, and an initiation
complex begins to build.
RNA polymerase
mRNA
3. RNA polymerase
associates with transcription
factors and DNA, this is the
initiation complex.
Transcription begins.
RNA Synthesis: Elongation, Transcription Bubble,
Termination
Transcription Bubble
1. Initiation ! Unwinding at Promoter site
2. Elongation ! Pairing of RNA nucleotides added to RNA 3’ end
3. Termination ! Stop Sequence!!!!!!!!!!
DNA Coding
strand
5’
GA
T
T
A C
TA
3’
3’
GA
C
T
T
3’
A
G C A U C G U
C
T
G T A G C A
A
C
AGT
C T
G
5’
RNA
5'
DNA Template strand
RNA polymerase
RNA-DNA hybrid helix
22
IV. Posttranscriptional Modifications
mRNA is processed in Eukaryotic Cells
1) Addition of a 5’ cap
2) Addition of a 3’ poly-A tail
3) Introns are Spliced from the transcript
This represents a major difference with
Prokaryotic cells, which have no
posttranscriptional modification
IV. Posttranscriptional Modifications
1) Addition of a 5’ cap: A modified Guanosine nucleotide
Eukaryotic mRNA Processing
protects against degradation; translation initiation
2) Addition of a 3’ poly-A tail: many Adenosine
nucleotides, also for protection
5' cap
HO OH
CH2
N+
Methyl group
CH3
5’ CAP
3’ poly-A Tail
P
P P
mRNA
ail 3'
-A t
o ly
p
'
A
3
A
A A A
P
PP
5' G
CH3
23
3) Introns are Spliced from the transcript
Primary RNA
transcript
5’
3’
Cap
Exons
Introns
Poly-A
tail
mRNA
Introns are excised from the RNA transcript, and the
remaining exons are spliced together, producing mRNA.
A word on Alternative Splicing and the human genome
Exon
(coding region)
DNA
Primary
RNA
transcript
Intron
(noncoding region)
1
5' cap
2
3
4
5 6
Transcription
7
3' poly-A
tail
Introns are cut out and
coding regions are
spliced together
Mature mRNA transcript
24
V. Translation
5’
Cap
Small
ribosomal
subunit
Nuclear
pore
mRNA
3’
Large
ribosomal
subunit
Poly-A
tail
Cytoplasm
mRNA is transported out of the nucleus. In the cytoplasm,
ribosomal subunits bind to the mRNA.
V. Translation
Strand to be transcribed
DNA
T A C T
T C A A A A T C
A T G A A G T T T T A G
Transcription
mRNA synthesis
A U G A A G U U U U A G
Translation
Start
codon
codon
codon
Met
Lys
Phe
Protein synthesis
Stop
codon
25
V. Translation
A. mRNA, tRNA, rRNA leave nucleus
•
through nuclear pores
B. Preparation in cytoplasm:
mRNA is bound by ribosomes (rRNA and many proteins)
tRNA: 45 types – some tRNAs recognize more than one codon
20 specific enzymes in cytoplasm attach correct AA
to each tRNA
•
•
tRNA has anti-codon
anti-codons pair with complimentary codon on mRNA
Ribosomes
Large ribosomal
subunit
P site
E site
E = exit
P = peptidyl (peptide)
A site
E
P
A = aminoacyl
Small ribosomal
subunit
A
mRNA
binding
site
26
OH
3’
5’
Transfer RNA Structure (tRNA)
Amino acid
attaches here
Anticodon
Transfer RNA Structure (tRNA)
Activating Enzymes Attach Amino Acids to t-RNA
One activating enzyme for each of 20 amino acids = aminoacyltRNA synthetases
Trp C=O
O
Trp
H2O
tRNATrp
O
ACC
UGG
Anticodon
Tryptophan
attached to
tRNATrp
O
C=
Trp C=O
OH
OH
Activating
enzyme
mRNA
tRNATrp binds to
UGG codon of
mRNA
27
C. mRNA translated to amino acid sequence
1) Initiation complex forms
= tRNA with methionine & small
ribosomal subunit
2) Initiation complex binds to mRNA,
Pairs with AUG codon nucleotides
3) large ribosome subunit binds to
small ribosome subunit
• mRNA fits in groove
• Methionine tRNA fits into the
“P” binding site on large subunit
Protein Synthesis Initiation: Review
Leader
sequence
Large
ribosomal
subunit
Initiation
factor
fMet
N
fMet
E site
C
e
fM
A
G
AU t
tR
fMet
fMet P site
A site
mRNA
A
U
UAC
mRNA
A UG
Initiation
factor
UAC
AUG
5'
UAC
AUG
3'
Initiation
complex
Small ribosomal subunit
(containing ribosomal RNA)
28
4) Second tRNA enters the “A” site
Only one with correct anticodon will bind
Leu
tRNA
fMet
A
C
P site (occupied)
Elongation
factor
G
5'
E site
A site
U AC
GAA U
A UGCU
3'
mRNA
5) Catalytic site (“P”) catalyzes peptide bond
! growing protein transferred to incoming AA, as…
Peptide
Bond Forms
fMet
Leu
5'
U A C GAC
A U G C U G A AU
3'
29
5) Catalytic site (“P”) catalyzes peptide bond
! growing protein transferred to incoming AA, as…
! Ribosome moves down mRNA—TRANSLOCATION
fMet
Leu
5'
C
UAC GA G A A U
U
C
G
A U
3'
6) Empty tRNA released
• Original tRNA moves over to “E” site
• “A” site ready for next tRNA
fMet
Leu
5'
C
UAC GA G A A U
U
C
A U G
3'
30
Protein Synthesis: Elongation & Translocation
P site
Cytoplasm
tRNA
E site
A site
tRNAs bring their amino acids in at the A site on the ribosome.
Peptide bonds form between amino acids at the P site, and
tRNAs exit the ribosome from the E site.
Protein Synthesis Termination
• Stop Codon or Nonsense Codon
• No tRNA associated with Stop Codons ! no Translocation
• But, Stop Codons are recognized by Release Factors
Val
Ser
Ala
Polypeptide chain
released
Trp
tRNA
Release Factor
5'
AC C
UGG U A A
3'
31
Protein Synthesis Termination
Polypeptide chain
released
Val
Release
factor
Ser
Ala
ACC
UGGU AA
tRNA
Ala Trp
Trp
P site
E
site
5'
Val Ser
tRNA
A CC
A
site
3'
5'
A CC
UGGUAA
3'
mRNA
Summary and key points of transcription & translation:
a) genes code for proteins
Exception: genes coding for tRNA & rRNA
b) Transcription of DNA to complimentary mRNA
c) Enzymes in cytoplasm attach correct AA on to
tRNA
d) tRNA’s carry AA’s to ribosomes
e) Ribosomes link AA’s --> peptide bonds
f) Sequence of protein determined by base pairing,
protein sequence determines folding and function!
32
VI. Comparison of Gene Expression in Prokaryotes
and Eukaryotes
Bacterial chromosome
Eukaryotic Chromosome
DNA
Transcription
mRNA
Translation
Intron Transcription
Primary
RNA transcript
Processing
5’
3’
mRNA
Poly-A tail
Cap
Protein
Nuclear envelope
Translation
Protein
VII. Structure of a Eukaryotic Gene
33
VIII. Control of Gene Expression
If each cell has the same DNA, how do we get all
of the different cells in our bodies?
Nerve cells
Muscle cells
Bone cells
Differentiation: cells become specialized. Results
from selective gene expression—the turning on and
turning off of specific genes
VIII. Control of Gene Expression
A) Background
Proteins could be structural, catalytic, regulatory or
involved in defense, motion, storage, or transport
Genes code for proteins (remember our
exceptions)
Regulation of gene expression is critical :
Keep in mind:
• each human cell has complete genome (totipotent)
• gene expression can change over time
• many genes are never expressed in any one cell
34