Post on 14-Jan-2016
Genetics News• Fly lab preliminary report due next week
• James Sikorski (Searle Pharmaceuticals)
Speaks on the process of drug discovery Friday, 10 November, 1:30 PM W-201 Informal discussion with students afterwards
• Lab next week in Jepson?
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 promoterB. Role of positive-acting regulators on transcriptionC. 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• Starvation for N• Growth stops
All or none
• Presence of glucose• Starvation for N
Broad, Graded
• Presence of lactose• Presence of tryptophan
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
Prokaryotic
Regulation of transcription
ß-globin
Eukaryotic
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
0
0.5
1
1.5
2
2.5
3
3.5
4
1
Step 3: Mutagenize
Step 4: Return mutant region to ß-globin gene
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
0
1
2
3
4
1
Relative Transcription Level
-100 -80 -60 -40 -20 CAP
SQ3. Are most mutations in upstream region detrimental?SQ4. If this were lac, where would worst mutations be?
What regulates ß-globin transcription?Experimental dissection of regulatory region
Results
0
1
2
3
4
1
Relative Transcription Level
-100 -80 -60 -40 -20 CAP
SQ5. Where were the most detrimental mutations?
AGAGCGACACCCTGGTAAGGGCCAATCTGC. . .AGAGCATATAAGGT
Regulatory sequences for eukaryotic genes are complex
TATA box TATAAAACAAT box GCCCAATCTGC box GGGCGGOctamer ATTGCAT
Histone H2B gene upstream region
Thymidine kinase gene upstream region
What are these sites?
Sites are binding sites for regulatory proteins
SQ6. Why is TATA box position unimportant, CAAT box not?SQ7. How might repression work in eukaryotes?Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.
RNA Processing
DNA
AUG
3’mG5’ppp
AAAAAAAAAAAAAAAAAAA...
Capping
Polyadenylation
AAAAAAAAAAAAAAAAAAA...
Splicing
E E EII
E E E
II
Transcription
AAUAAA
Why???
Special problems of Eukaryotes
O
Linear search
Cooperative binding
bacterium
eukaryote
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 BacteriaEukaryotes use signals at a distance
SQ10. Examples?
Bacteria
Eukaryotes
Transcriptional regulation
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.
Eukaryotes vs BacteriaEukaryotes use signals at a distance
SQ10. Examples?Translation regulation
Bacteria
Eukaryotes
AAGGAGGnnnnnAUG
Eukaryotes vs BacteriaEukaryotes use signals at a distance
Effect on evolution
Hair gene
Toenail gene
Hair gene