L3 Transcription Initiation Regulation

Question 1

Double mutant effects

Answer 1

• effects same mechanism or different mechanism
• same mechanism: not additive
• different mechanism: additive

Question 2

Positive regulation of the lac operon

Answer 2

1. Local regulator, regulates only the lac operon, lacI repressor
2. Global regulator, regulates 100+ genes in E.coli, carbon metabolite repression (CAP protein)
   Both act independently on Lac operon
   - Glucose uptake inhibits lacY lac permease

Question 3

Regulation by CAP protein

Answer 3

• positive

- inducible by cAMP

Question 4

CYA

Answer 4

• codes for adenylate cyclase

- adenylate cyclase converts ATP to cAMP

Question 5

CAP protein

Answer 5

• function as dimer
• has separate DNA binding domain and activation domain
• interacts with CTD of alpha subunit of RNA pol
• can activate many promoters from different relative positions. flexible
• cap binding can bend DNA, allows interaction with with alpha subunit of RNAP to initiate transcription

Question 6

Evidence for CAP-RNAP interaction

Answer 6

• biochemical studies

- genetic studies

Question 7

Genetic identification of CAP binding site - mutants

Answer 7

lac- mutants that map to promoter region, not in lacZ region

• class 1: mutant L8
• class 2: mutant L157
• L8 and L157 mutants are in different regions

Question 8

Class 2 mutant L157

Answer 8

different mechanism to CAP

Question 9

Class 1 mutant L8

Answer 9

same mechanism as CAP

Question 10

Class 1 mutant L8

Answer 10

same mechanism as CAP

Question 11

Arabinose operon

Answer 11

2 positive regulators:

1. CAP protein
2. AraC
   - AraC can bind to araI1, araI2 and araO2

AraC is always bound to DNA but the way it binds differ depending on whether arabinose is present or not, its conformational change affects its binding
Conformational change causes AraC to recognise different sites; the three binding sites of AraC are not identical

Question 12

MerR

Answer 12

• merR: positive regulator of merT, confers resistance to mercury
• structural gene: merT

Question 13

Evidence for CAP-RNAP interaction - Genetic studies

Answer 13

CAP “activation-negative” mutants

RNAP alpha-subunit mutants

Question 14

Cap mutants

Answer 14

cap-

cya-