class i mutants class ii mutants class iii mutants class i ......dna rna polymerase 5! 3! 5! 3! 1...
TRANSCRIPT
RESULTS
EXPERIMENT
CONCLUSION
Growth: Wild-type cells growing and dividing
No growth: Mutant cells cannot grow and divide
Minimal medium
Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants
Minimal medium (MM) (control) MM + ornithine
MM + citrulline
Co
nd
itio
n
MM + arginine (control)
Class I mutants (mutation in
gene A) Wild type Class II mutants
(mutation in gene B)
Class III mutants (mutation in
gene C)
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor Enzyme A Enzyme A Enzyme A Enzyme A
Enzyme B
Ornithine Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B
Citrulline Citrulline Citrulline Citrulline Enzyme C Enzyme C Enzyme C Enzyme C
Arginine Arginine Arginine Arginine
1
TRANSCRIPTION
TRANSLATION
DNA
mRNA Ribosome
Polypeptide
(a) Bacterial cell
Nuclear envelope
TRANSCRIPTION
RNA PROCESSING Pre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
2
DNA molecule
Gene 1
Gene 2
Gene 3
DNA template strand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
3
Second mRNA base
Fir
st m
RN
A b
ase
(5ʹ′ e
nd
of
cod
on
)
Th
ird
mR
NA
bas
e (3ʹ′ e
nd
of
cod
on
)
4
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
5
Promoter Transcription unit
Start point DNA
RNA polymerase
5ʹ′ 5ʹ′ 3ʹ′ 3ʹ′
Initiation 1
2
3
5ʹ′ 5ʹ′ 3ʹ′ 3ʹ′
Unwound DNA
RNA transcript
Template strand of DNA
Elongation
Rewound DNA
5ʹ′
5ʹ′ 5ʹ′
5ʹ′
5ʹ′
3ʹ′ 3ʹ′
3ʹ′
3ʹ′
RNA transcript
Termination
5ʹ′ 5ʹ′ 3ʹ′ 3ʹ′
3ʹ′ 5ʹ′ Completed RNA transcript
Newly made RNA
Template strand of DNA
Direction of transcription (“downstream”)
3ʹ′ end
RNA polymerase
RNA nucleotides
Nontemplate strand of DNA
Elongation
6
A eukaryotic promoter includes a TATA box
3ʹ′
1
2
3
Promoter
TATA box Start point
Template
Template DNA strand
5ʹ′ 3ʹ′ 5ʹ′
Transcription factors
Several transcription factors must bind to the DNA before RNA polymerase II can do so.
5ʹ′ 5ʹ′ 3ʹ′ 3ʹ′
Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex.
RNA polymerase II Transcription factors
5ʹ′ 5ʹ′ 5ʹ′ 3ʹ′
3ʹ′
RNA transcript
Transcription initiation complex
7
Protein-coding segment Polyadenylation signal 3ʹ′
3ʹ′ UTR 5ʹ′ UTR
5ʹ′
5ʹ′ Cap Start codon Stop codon Poly-A tail
G P P P AAUAAA AAA AAA …
8
Pre-mRNA
mRNA
Coding segment
Introns cut out and exons spliced together
5ʹ′ Cap
Exon Intron 5ʹ′
1 30 31 104
Exon Intron
105
Exon
146
3ʹ′ Poly-A tail
Poly-A tail 5ʹ′ Cap
5ʹ′ UTR 3ʹ′ UTR 1 146
9
RNA transcript (pre-mRNA)
Exon 1 Exon 2 Intron
Protein snRNA
snRNPs
Other proteins
5ʹ′
5ʹ′
Spliceosome
Spliceosome components
Cut-out intron mRNA
Exon 1 Exon 2 5ʹ′
10
Gene DNA
Exon 1 Exon 2 Exon 3 Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide
11
Polypeptide
Ribosome
Amino acids
tRNA with amino acid attached
tRNA
Anticodon
Trp
Phe Gly
Codons 3ʹ′ 5ʹ′
mRNA 12
Amino acid attachment site
3ʹ′
5ʹ′
Hydrogen bonds
Anticodon
(a) Two-dimensional structure
Amino acid attachment site
5ʹ′
3ʹ′
Hydrogen bonds
3ʹ′ 5ʹ′ Anticodon Anticodon
(c) Symbol used in this book (b) Three-dimensional structure
13
Amino acid Aminoacyl-tRNA synthetase (enzyme)
ATP
Adenosine P P P
Adenosine P
P P i
P P i
i
tRNA
tRNA
Aminoacyl-tRNA synthetase
Computer model
AMP Adenosine P
Aminoacyl-tRNA (“charged tRNA”)
14
Growing polypeptide Exit tunnel
Large subunit
Small subunit
tRNA molecules
E P A
mRNA 5ʹ′ 3ʹ′
(a) Computer model of functioning ribosome
P site (Peptidyl-tRNA binding site)
E site (Exit site)
A site (Aminoacyl- tRNA binding site)
E P A Large subunit
mRNA binding site
Small subunit
(b) Schematic model showing binding sites
Amino end Growing polypeptide
Next amino acid to be added to polypeptide chain
mRNA tRNA E
3ʹ′
5ʹ′ Codons
(c) Schematic model with mRNA and tRNA
15
3ʹ′ 3ʹ′ 5ʹ′ 5ʹ′ U
U A
A C G Met
GTP GDP Initiator
tRNA
mRNA
5ʹ′ 3ʹ′ Start codon
mRNA binding site
Small ribosomal subunit
5ʹ′
P site
Translation initiation complex
3ʹ′
E A
Met
Large ribosomal subunit
16
Amino end of polypeptide
mRNA
5ʹ′
3ʹ′ E
P site
A site
GTP
GDP
E
P A
E
P A
GDP
GTP
Ribosome ready for next aminoacyl tRNA
E
P A
17
Release factor
3ʹ′
5ʹ′ Stop codon (UAG, UAA, or UGA)
5ʹ′
3ʹ′
2
Free polypeptide
2 GDP
GTP
5ʹ′
3ʹ′
18
Growing polypeptides
Completed polypeptide
Incoming ribosomal subunits
Start of mRNA (5ʹ′ end)
Polyribosome End of mRNA (3ʹ′ end)
(a)
Ribosomes
mRNA
(b) 0.1 µm
19
Ribosome
mRNA
Signal peptide
Signal- recognition particle (SRP)
CYTOSOL Translocation complex
SRP receptor protein
ER LUMEN
Signal peptide removed
ER membrane
Protein
20
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
3ʹ′ 3ʹ′
3ʹ′
3ʹ′
3ʹ′
3ʹ′
5ʹ′ 5ʹ′
5ʹ′
5ʹ′ 5ʹ′
5ʹ′
C C T T T T G G A A A A
A A A G G U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
21
Wild-type
3ʹ′ DNA template strand 5ʹ′
5ʹ′
5ʹ′ 3ʹ′
3ʹ′
Stop
Carboxyl end Amino end
Protein
mRNA
3ʹ′ 3ʹ′
3ʹ′
5ʹ′ 5ʹ′
5ʹ′
A instead of G
U instead of C
Silent (no effect on amino acid sequence)
Stop
T instead of C
3ʹ′ 3ʹ′
3ʹ′
5ʹ′ 5ʹ′
5ʹ′
A instead of G
Stop
Missense
A instead of T
U instead of A
3ʹ′ 3ʹ′
3ʹ′
5ʹ′
5ʹ′
5ʹ′
Stop
Nonsense No frameshift, but one amino acid missing (3 base-pair deletion)
Frameshift causing extensive missense (1 base-pair deletion)
Frameshift causing immediate nonsense (1 base-pair insertion)
5ʹ′
5ʹ′
5ʹ′ 3ʹ′ 3ʹ′
3ʹ′
Stop
missing
missing
3ʹ′
3ʹ′
3ʹ′
5ʹ′
5ʹ′ 5ʹ′
missing
missing
Stop
5ʹ′
5ʹ′ 5ʹ′ 3ʹ′ 3ʹ′
3ʹ′
Extra U
Extra A
(a) Base-pair substitution (b) Base-pair insertion or deletion
22
RNA polymerase
DNA
Polyribosome
mRNA
0.25 µm Direction of transcription
DNA
RNA polymerase
Polyribosome
Polypeptide (amino end)
Ribosome
mRNA (5ʹ′ end)
23
TRANSCRIPTION
RNA PROCESSING
DNA
RNA transcript
3ʹ′
5ʹ′ RNA polymerase
Poly-A
Poly-A
RNA transcript (pre-mRNA)
Intron
Exon
NUCLEUS
Aminoacyl-tRNA synthetase
AMINO ACID ACTIVATION Amino acid
tRNA CYTOPLASM
Poly-A
Growing polypeptide
3ʹ′
Activated amino acid
mRNA
TRANSLATION
Cap
Ribosomal subunits
Cap
5ʹ′
E
P
A
A Anticodon
Ribosome
Codon
E
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