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