L2 Prokaryotic Regulation

Question 1

Phases of Transcription

Answer 1

1,2,3,4,5

Question 2

RNA Polymerase Structure

Answer 2

ABCD Z

Question 3

Other sigma factors

Answer 3

unusual circumstances e.g. environmental stresses

Question 4

Identifying functionally important DNA sequences that direct RNAPol to initiate only at promoters

Answer 4

1. comparative approach - consensus sequence
2. genetic approach - isolate mutations affecting promoter function, “weak” and “strong” promoters
3. biochemical approach - gel mobility shift assay, DNA modification protection assay

Question 5

DNA modification protection

Answer 5

• DNAse1

- immunoprecipitate

Question 6

Positive vs Negative

Answer 6

Positive Regulation

The binding of specific protein (activator) is required for transcription to begin.

DNA bound activators can regulate transcription by helping with ignition. To do this they sometimes tether RNA polymerase to the promoter.

Negative Regulation

The binding of a specific protein (repressor) inhibits transcription from occurring.

DNA bound repressors often act to prevent RNA polymerase from binding to the promoter, or by blocking the movement of RNA polymerase

Question 7

Inducible vs Repressible

Answer 7

Inducible system - the effector molecule interacts with the repressor protein such that it can not bind to the operator

Repressible system - the effector molecule interacts with the repressor protein such that it can bind to the operator

Question 8

Lac Operator

Answer 8

• dyad
• O1, O2, O3
• binds to each operator as a dimer
• repressor functions best as a tetramer

Question 9

Trp Operon

Answer 9

• negative
• repressible
• operator has dyad symmetry

Question 10

TrpR

Answer 10

• TrpR binds as a dimer, each monomer can bind to tryptophan to convert TrpR to an active repressor
• TrpR negatively regulates several different promoters: sequences in common but not identical
• all operators overlap promoter but relative distance from -10 and -35 sequence differ

Question 11

Lac operon mutants

Answer 11

See onenote summary page

Question 12

LacI repressor

Answer 12

helix-turn-helix domain, in major groove of DNA

binds as dimer

Question 13

Lac repressor binding

Answer 13

O1, O2, O3

O2 & O3 mutants

Question 14

Repressor interacts with RNA pol

Answer 14

• increase RNAP binding to lac promoter while preventing transcription initiation
• Means that RNA pol is there ready to go as soon as allolactose is present, a way to make sure that transcription occurs as rapidly as possible when the inducer is present