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Genetics News
• Fly lab preliminary report due next week
• Lab next week in Jepson?
• James Sikorski (Searle Pharmaceuticals)
Speaks on the process of drug discovery
Friday, 10 November, 1:30 PM W-201
Informal discussion with students afterwards
Topics
I. Large scale transcriptional regulation in prokaryotes (pp.342-343)
II. Transcriptional regulation in eukaryotes (pp.345-350)
A. EXPERIMENT: Fine structure of -globin promoter
B. Role of positive-acting regulators on transcription
C. RNA processing
III. Comparison between prokaryotes and eukaryotes
Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
Modes of regulation (prokaryote)

Promoter choice
by alternative
sigma factors
Increased transcription
by positive regulators
Decreased transcription
by negative regulators
(repressors)
SQ1. Which mutation affects expression of more genes:
- in a gene encoding a repressor?
- in a gene encoding a sigma factor?
Modes of regulation (prokaryote)

Promoter choice
by alternative
sigma factors
Increased transcription
by positive regulators
Decreased transcription
by negative regulators
(repressors)
• Heat shock
• Presence of glucose
• Starvation for N
• Presence of lactose
• Presence of tryptophan
• Starvation for N
• Growth stops
All or none
Broad, Graded
Specific, graded
Modes of regulation (prokaryote)

Promoter choice
by alternative
sigma factors
Increased transcription
by positive regulators
Decreased transcription
by negative regulators
(repressors)
SQ2. Regulation by alternative sigma factor vs a repressor?
Phosphate starvation
phosphate transporter
Regulation of transcription
Prokaryotic
Eukaryotic
ß-globin
?
?
Regulation
Goal
Find sequences necessary for
transcription of ß-globin
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Mutagenesis
Step 1,2: Clone region
Step 3: Mutagenize
Step 4: Return mutant
region to ß-globin gene
4
3.5
3
2.5
2
1.5
1
0.5
0
1
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Expression
Step 5: Put construct in cells
Step 6: Allow expression
Step 7: Isolate RNA
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Quantitation (part I)
Step 8: Make probe
Step 9: Mix probe + RNA
Step 10: Digest unbound
RNA and probe
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Quantitation (part II)
Step 11: Apply samples
to gel
Step 10: Blot gel, expose
X-ray film
Step 11: Quantitate degree
of exposure
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Results
4
3
Relative
Transcription
Level
2
1
0
1
-100
-80
-60
-40
-20
CAP
SQ3. Are
SQ4.
If this
most
were
mutations
lac, where
in upstream
would worst
region
mutations
detrimental?
be?
What regulates ß-globin transcription?
Experimental dissection of regulatory region
Results
4
3
Relative
Transcription
Level
2
1
0
1
-100
AGA
-80
-60
-40
-20
CAP
GCGACACCCTGGTAAGGGCCAATCTGC. . .AGAGCATATAAGGT
SQ5. Where were the most detrimental mutations?
Regulatory sequences for
eukaryotic genes are complex
Histone H2B gene upstream region
Thymidine kinase gene upstream region
TATA box
CAAT box
GC box
Octamer
TATAAAA
GCCCAATCT
GGGCGG
ATTGCAT
What are these sites?
Sites are binding sites for regulatory proteins
SQ6. Why
SQ7.
How is
might
TATA
repression
box position
workunimportant,
in eukaryotes?
CAAT box not?
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
RNA Processing
DNA
Transcription
3’mG5’ppp
Capping
AUG
AAUAAA
Polyadenylation
E
Splicing
I E
I E
AAAAAAAAAAAAAAAAAAA...
I
I
E
E
E
AAAAAAAAAAAAAAAAAAA...
Why???
Special problems of Eukaryotes
eukaryote
bacterium
Cooperative binding
Linear search
O
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
Special problems of Eukaryotes
Eukaryotes: Homogeneous in DNA, heterogeneous in form
Eukaryotes vs Bacteria
Eukaryotes use signals at a distance
SQ10. Examples?
Transcriptional regulation
Bacteria

Eukaryotes
Figure from Griffiths et al (1996) Introduction to Genetic
Analysis, 6th ed., WH Freeman and Co.
Eukaryotes vs Bacteria
Eukaryotes use signals at a distance
SQ10. Examples?
Translation regulation
Bacteria
AAGGAGGnnnnnAUG
Eukaryotes
Eukaryotes vs Bacteria
Eukaryotes use signals at a distance
Effect on evolution
Hair gene
Toenail gene
Hair gene