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Experimental Procedure
No growth
on minimal
medium
Growth on
minimal
medium
plus arginine
Wild-type
Neurospora
crassa
Mutagenize
with X-rays
Grow on
rich medium
arg mutants
Results
Mutation
in Enzyme
Plus
Ornithine
Plus
Plus
Plus
Citruline Arginosuccinate Arginine
E
F
G
H
Conclusion
Glutamate
Enzymes
encoded
by arg
genes
arg
genes
Ornithine
Citruline
Arginosuccinate
Arginine
E
F
G
H
arg E
arg F
arg G
arg H
1
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Prokaryotes
5´
DN A
template
strand
Eukaryotes
C
T
mRNA
3´
Transcription
3´
G
A
Translation
5´
Protein
2
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Replication
DNA
Transcription
Reverse transcription
RNA
Translation
Protein
3
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Sentence with Spaces
WHY DID THE RED BAT EAT THE FAT RAT
Delete one letter
WHY DID HE RED BAT EAT THE FAT RAT
Only one word changed
Sentence with No Spaces
WHYDIDTHEREDBATEATTHEFATRAT
Delete one letter
WHYDIDHEREDBATEATTHEFATRAT
All words after deletion changed
4
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SCIENTIFIC THINKING
Hypothesis: The genetic code is read in groups of three bases.
Prediction: If the genetic code is read in groups of three, then a
deletion of one or two bases would cause drastic changes to the
encoded protein. Deletion of three bases, however, could produce
a protein close to the “normal” sequence.
Test: Single-base deletion mutants are collected, each of which
exhibits a mutant phenotype. Three of these deletions in a single
region are combined to assess the effect of deletion of three bases.
one Bases Deleted
Met Pro Thr His Arg Asp Ala Ser
Amino acids
AUGCCUACGCACCGCGACGCAUCA
Delete one bases
AUGCCUAGCACCGCGACGCAUCA
All amino acids changed
after deletion
Met Pro Ser Thr Ala Thr His
Three Bases Deleted
Met Pro Thr His Arg Asp Ala Ser
Amino acids
AUGCCUACGCACCGCGACGCAUCA
Delete three bases
AUGCCUCACCGCGACGCAUCA
Met Pro His Arg Asp Ala Ser
Amino acids do not
change after third deletion
Result: The combination of three deletions does not have the same
drastic effect as the loss of one or two bases.
Conclusion: The genetic code is read in groups of three.
Further Experiments: If you also had mutants with one base
additions, what would be the effect of combining a deletion and an
addition?
5
SCIENTIFIC THINKING
6
SCIENTIFIC THINKING
7
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RNA
polymerase
DNA
Start site
Unwinding
Coding strand
Rewinding
‫׳‬3
‫׳‬3
‫׳‬5
‫׳‬5
Downstream
‫׳‬3
Upstream
mRNA
‫׳‬5
Template strand
Transcription bubble
8
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RNA polymerase
DNA and RNA
polymerase dissociates
DNA
mRNA
dissociates
from DNA
‫׳‬3
‫׳‬5
‫׳‬5
‫׳‬3
Four, or more U
ribonucleotides
Cytosine
mRNA hairpin
causes RNA
polymerase to pause
Guanine
Adenine
Uracil
‫׳‬5
9
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0.25µm
RNA polymerase
DNA
Polyribosome
mRNA
Polypeptide
chains
Ribosomes
© Dr. Oscar Miller
10
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Other transcription factors
RNA polymerase II
Eukaryotic
DNA
Transcription
factor
TATA box
Initiation
complex
11
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5´ cap
HO
OH
P
CH2
N+
CH3
P
P
+
3´
Methyl group
P
5´
P
P
mRNA
CH3
12
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E1
I1
E2
I2
E3
I3
DNA template
‫׳‬5
poly-A tail ‫׳‬3
Primary RNA transcript
Introns are removed
p
c ‫׳‬5
a.
I4
Exons
Introns
Transcription
c
E4
‫׳‬3
Mature mRNA
Intron
1
mRNA
3
2
4
DNA
7
5
6
Exon
b.
c.
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
13
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snRNA
Exon 1
snRNPs
Intron
Exon 2
A
5´
3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome
A
5´
3´
2. snRNPs associate with other factors to form spliceosome.
Lariat
A
5´
3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut.
Exon 1
5´
Excised
intron
Exon 2
Mature mRNA
3´
4. Exons are joined; spliceosome disassembles.
14
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2D “Cloverleaf” Model
Acceptor end
‫׳‬3
3D Ribbon-like Model
Acceptor end
3D Space-filled Model
Acceptor end
Icon
Acceptor end
‫׳‬5
Anticodon
loop
Anticodon loop
Anticodon loop
Anticodon end
c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by
John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is fi nanced by grant GM63208
15
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Amino
group
NH3+
ATP
Pi Pi
tRNA
site
Carboxyl
group
Trp
C
Charged tRNA travels to ribosome
NH
O
O–
Amino
acid site
3
Accepting
site
+
Trp
AM
C
O
Trp
NH3+
Trp
P O
OH
C
O
O
A MP
tRNA
Aminoacyl-tRNA
Anticodon
synthetase
specific to tryptophan
Charged
tRNA
dissociates
16
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Large
subunit
3´
Small
subunit
Large
subunit
90°
Small
subunit
Large
subunit
0°
mRNA
Small
subunit
5´
17
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fMet
E site
AUG
U A
C
tRNA in
P site
U A C
A U G
Large
subunit
3´
Initiation
factor
mRNA
A site
3´
3´
3´
Initiation
factor
5´
Small
subunit
5´
GTP
GDP
+
5´
5´
Pi
Initiation complex
Complete ribosome
18
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NH3+
O
NH3+
O
C
O
C
O
NH2
Amino
acid 1
C
Peptide
bond
N
Amino
acid 2
Amino
acid 2
Amino
acid 2
Amino
acid 1
3´
Polypeptide
chain
NH3+
Amino
group
Amino
acid 1
Amino end
(N terminus)
C
O
Peptide
bond
formation
“Empty”
tRNA
OH
Amino
acid 3
O
Amino
acid 4
O
Amino
acid 5
Amino
acid 6
Amino
acid 7
5´
COO–
A site
P site
Carboxyl end
(C terminus)
19
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3´
GDP
+
Pi
Elongation
factor
E
Elongation
factor
GTP
P
A
5´
3´
3´
E
P
A
E
5´
A
P
5´
Sectioned ribosome
GTP
GTP
Next round
Elongation
factor
Growing
polypeptide
“Ejected” tRNA
3´
3´
E
P
Elongation
factor
GDP + Pi
A
E
P
A
5´
5´
20
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Polypeptide
chain releases
Dissociation
3´
Release
factor
5´
3´
5´
Sectioned
ribosome
C
A C G
U G
E
A
U A
A
P
21
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Rough endoplasmic
reticulum (RER)
Cytoplasm
Lumen of the RER
Protein channel
SRP binds to signal Docking
peptide, arresting
elongation
Signal recognition
particle (SRP)
NH22
NH
Polypeptide
elongation
continues
Signal
Exit tunnel
Ribosome
synthesizing
peptide
22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Primary RNA transcript
Poly-A tail
Cut intron
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Cytoplasm
Amino acids
tRNA arrivesin A site
3´
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Empty tRNA moves into
E site and is ejected
Lengthening
polypeptide chain
Emptyt
RNA
Mature mRNA
5´ cap
3´
3´
mRNA
5´
A site
P site
E site
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
5´
5. Peptide bonds form between the
amino terminus of the next amino
acid and the carboxyl terminus of
the growing peptide. This transfers
the growing peptide to the tRNA in
the A site, leaving the tRNA in the
P site empty.
5´
6. Ribosome translocation moves the
ribosome relative to the mRNA and
its bound tRNAs. This moves the
growing chain into the P site, leaving
the empty tRNA in the E site and the
A site ready to bind the next
charged tRNA.
23
24
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Normal
Deoxygenated
Tetramer
Normal HBB Sequence
Polar
Leu
C
T
Thr
G
A
C
Pro
T
C
C
Glu
T
G
A
Glu
G
A
A
Lys
G
A
A
Ser
G
T
C
Amino acids
T Nucleotides
Abnormal
Deoxygenated
Tetramer
α1
α2
α1 α2
β1
β2
β1 β2
Hemoglobin
tetramer
"Sticky" nonpolar sites
Abormal HBB Sequence
Nonpolar (hydrophobic)
Leu
C
T
Thr
G
A
C
val
Pro
T
C
C
T
G
T
Glu
G
A
A
Lys
G
A
A
Ser
G
T
C
Amino acids
T Nucleotides
Tetramers form long chains
when deoxygenated. This
distorts the normal red blood
cell shape into a sickle shape.
25
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C
G
A A
T T
Coding
5´–ATGCCTTATCGCTGA–3´
Template
3´–TACGGAATAGCGACT–5´
mRNA
5´–AUGCCUUAUCGCUGA–3´
Protein
Met Pro Thr Arg Stop
a.
Silent Mutation
C
G
Coding
5´–ATGCCCTATCGCTGA–3´
Template
3´–TACGGGATAGCGACT–5´
mRNA
5´–AUGCCCUAUCGCUGA–3´
Protein
Met Pro Thr Arg Stop
b.
Missense Mutation
A
T
Coding
5´–ATGCCCTATCACTGA–3´
Template
3´–TACGGGATAGTGACT–5´
mRNA
5´–AUGCCCUAUCACUGA–3´
Protein
Met Pro Thr His Stop
c.
Nonsense Mutation
A
T
Coding
5´–ATGCCCTAACGCTGA–3´
Template
3´–TACGGGATTGCGACT–5´
mRNA
5´–AUGCCCUAACGCUGA–3´
Protein
d.
Met Pro Stop
26
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Deletion
Deleted
AB C D E F G H I J
AE F G H I J
a.
Duplication
Duplicated
A B C D E F G H I J
A B C D B C D E F G H I J
b.
Inversion
Inverted
AB C D E F G H I J
AD C B E F G H I J
c.
Reciprocal Translocation
d.
AB C D E F G H I J
K L M D E F G H I J
KL M N O P Q R
A B C N OP Q R
27