Download Replication Reverse transcription Protein Transcription Translation

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Experimental Procedure
No growth
on minimal
medium
Prokaryotes
Growth on
minimal
medium
plus arginine
Wild-type
Neurospora
crassa
Mutagenize
with X-rays
Grow on
rich medium
arg mutants
5´
Results
Mutation
in Enzyme
Plus
Ornithine
Eukaryotes
Plus
Plus
Plus
Citruline Arginosuccinate Arginine
C
E
T
C
T
DN A
template
strand
A
Transcription
3´
G
F
G
U
G
mRNA
3´
A
C
H
A
G
Translation
Conclusion
Glutamate
Enzymes
encoded
by arg
genes
arg
genes
Ornithine
Citruline
Arginosuccinate
5´
Arginine
E
F
G
H
arg E
arg F
arg G
arg H
Protein
1
2
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Sentence with Spaces
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WHY DID THE RED BAT EAT THE FAT RAT
Delete one letter
Replication
WHY DID HE RED BAT EAT THE FAT RAT
DNA
Transcription
Only one word changed
Reverse transcription
RNA
Translation
Sentence with No Spaces
WHYDIDTHEREDBATEATTHEFATRAT
Protein
Delete one letter
WHYDIDHEREDBATEATTHEFATRAT
All words after deletion changed
3
4
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SCIENTIFIC THINKING
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
6
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SCIENTIFIC THINKING
RNA
polymerase
DNA
Start site
Unwinding
Coding strand
Rewinding
‫׳‬3
‫׳‬3
‫׳‬5
‫׳‬5
Downstream
‫׳‬3
Upstream
Template strand
mRNA
Transcription bubble
‫׳‬5
7
8
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RNA polymerase
DNA and RNA
polymerase dissociates
DNA
mRNA
dissociates
from DNA
‫׳‬3
0.25µm
‫׳‬5
RNA polymerase
‫׳‬5
‫׳‬3
DNA
Four, or more U
ribonucleotides
mRNA hairpin
causes RNA
polymerase to pause
Polyribosome
mRNA
Cytosine
Guanine
Adenine
Uracil
Polypeptide
chains
Ribosomes
‫׳‬5
© Dr. Oscar Miller
9
10
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5´ cap
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Other transcription factors
HO
RNA polymerase II
OH
P
CH2
Eukaryotic
DNA
Transcription
factor
N+
CH3
TATA box
+
3´
G
5´
A
lypo
l
tai
A
AA
Methyl group
Initiation
complex
11
P
P
P
P
P
AA
3´
AA
mRNA
CH3
12
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E1
I1
E2
I2
E3
I3
E4
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I4
snRNA
Exon 1
snRNPs
Intron
Exon 2
A
5´
DNA template
Exons
Introns
Transcription
c
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
‫׳‬5
poly-A tail ‫׳‬3
Spliceosome
Primary RNA transcript
Introns are removed
p
c ‫׳‬5
a.
3´
Branch point A
A
5´
‫׳‬3
3´
2. snRNPs associate with other factors to form spliceosome.
Mature mRNA
Lariat
A
Intron
5´
1
mRNA
3
2
4
DNA
7
5
6
Exon 1
Exon
b.
5´
c.
Acceptor end
‫׳‬3
3D Ribbon-like Model
3D Space-filled Model
Acceptor end
Acceptor end
14
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Amino
group
Icon
NH3+
Acceptor end
ATP
Pi Pi
Carboxyl
group
Trp
C
Charged tRNA travels to ribosome
NH
O
3
O–
Accepting
site
Amino
acid site
Anticodon loop
NH
+
3
Trp
C
AM
+
O
Trp
AM
P
P O
OH
C
NH
O
O
3
+
NH3+
Trp
C
AMP
OH
Trp
C
O
O
O
O
tRNA
tRNA
site
Anticodon loop
3´
13
‫׳‬5
Anticodon
loop
Excised
intron
Exon 2
Mature mRNA
4. Exons are joined; spliceosome disassembles.
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
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2D “Cloverleaf” Model
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.
Charged
tRNA
dissociates
Aminoacyl-tRNA
Anticodon
synthetase
specific to tryptophan
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
16
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Large
subunit
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3´
90°
fMet
Small
subunit
Large
subunit
Small
subunit
Large
subunit
0°
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
mRNA
Small
subunit
5´
17
18
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3´
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GDP
NH3+
O
O
C
C
O
O
Peptide
bond
formation
GTP
Amino
acid 4
Amino
acid 5
P
E
A
E
A
P
5´
Sectioned ribosome
GTP
GTP
Next round
Elongation
factor
3´
3´
Carboxyl end
(C terminus)
E
P
Elongation
factor
GDP + Pi
Growing
polypeptide
“Ejected” tRNA
Amino
acid 7
COO–
A site
P site
3´
5´
Amino
acid 6
5´
A
P
5´
3´
Amino
acid 3
O
O
OH
E
Elongation
factor
Amino
acid 2
Amino
acid 2
“Empty”
tRNA
Elongation
factor
Amino
acid 1
Peptide
bond
C
O
Pi
NH2
C
N
Amino
acid 2
Amino
acid 1
3´
Polypeptide
chain
NH3+
NH3+
Amino end
(N terminus)
Amino
group
Amino
acid 1
+
A
E
P
A
5´
5´
19
20
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Rough endoplasmic
reticulum (RER)
Polypeptide
chain releases
Cytoplasm
Lumen of the RER
Dissociation
Protein channel
3´
Release
factor
SRP binds to signal Docking
peptide, arresting
elongation
Signal recognition
particle (SRP)
5´
3´
NH22
NH
Polypeptide
elongation
continues
Signal
Exit tunnel
Ribosome
synthesizing
peptide
5´
Sectioned
ribosome
C
A C G
U G
A
U A
A
P
E
21
22
23
24
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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.
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C
G
5´–ATGCCTTATCGCTGA–3´
Template
3´–TACGGAATAGCGACT–5´
mRNA
5´–AUGCCUUAUCGCUGA–3´
Protein
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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
Met Pro Thr Arg Stop
a.
Normal
Deoxygenated
Tetramer
Normal HBB Sequence
A A
T T
Coding
α1
α2
β1
β2
Abnormal
Deoxygenated
Tetramer
Silent Mutation
C
G
α1 α2
Coding
5´–ATGCCCTATCGCTGA–3´
Template
3´–TACGGGATAGCGACT–5´
β1 β2
mRNA
5´–AUGCCCUAUCGCUGA–3´
Protein
Hemoglobin
tetramer
"Sticky" nonpolar sites
Met Pro Thr Arg Stop
b.
Abormal HBB Sequence
Missense Mutation
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
A
T
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.
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
25
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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
d.
Met Pro Stop
26