positive regulation of the lactose operon

unlike the trp operon, the lac operon is under both (?) transcription controls

positive and negative

component of positive lac operon regulation

1. Lac promoter -> relatively weak

2. CAP -> activator protein needed to help RNA polymerase bind to the promoter

• It binds to DNA near the promoter

1. cAMP -> messenger molecule -> CAP must bind to cAMP before CAP (CAP-cAMP dimer) can bind to DNA

what does CAP stand for?

catabolite activator protein

(?) is the preferred sugar of E. coli

glucose

as long as glucose is present, the lac operon is switched (?)

off

cAMP responds to changes in (?) concentration

glucose

there is an (?) relationship between glucose levels and cAMP concentration

inverse

what happens when glucose concentration falls?

1. cAMP concentration rises

2. levels of CAP-cAMP dimer also rise (more cAMP available to bind to CAP)

3. CAP-cAMP dimer can bind to the lac operon DNA and activate transcription

why is the previous mechanism positive regulation of the lac operon

because there is an activator (CAP-cAMP protein), not a repressor protein, which increases DNA binding which activates transcription

what happens after cAMP binds to CAP

1. cAMP binds to CAP protein

2. causes change in shape which allows CAP to bind to the CAP site

3. the bound CAP-cAMP dimer interacts with RNA polymerase

4. activates transcription -> greatly increases the rate of transcription initiation

the operon is highly expressed only when (a) is present and (b) is absent

(a) lactose

(b) glucose

(?) binding turns on lac operon in positive regulation

CAP

Explain what happens when:

1. When both glucose and lactose are present

 2. When glucose is present and lactose is absent

 3. When both are absent

4. When lactose is present and glucose is absent

1. CAP is not bound → low expression of lac operon

2. Repressor blocks transcription → CAP is not bound

3. CAP is bound but repressor blocks transcription

4. CAP is bound and repressor is not bound → transcription activated

1. Glucose is present and lactose is absent

2. Glucose is present and lactose is present

3. Glucose is absent and lactose is present

1. Glucose is present and lactose is absent

Operon is off -> repressor bound

Low levels of transcription

 

2. Glucose is present and lactose is present

Allolactose formed -> inducer

Operon is on

Low levels of transcription

3. Glucose is absent and lactose is present

CAP-cAMP dimer bound

No repressor bound

Operon is on

High levels of transcription

LacI is a (?) protein

repressor

allolactose binds to (a) to remove it from the DNA and prevent transcription, therefore allolactose is an (b)

(a) lacI

(b) inducer

CAP is an (?) protein

activator

cAMP is an (?)

inducer

cAMP binds to CAP, forming a cAMP-CAP dimer which binds to the lac operon and (?) transcription

activates

complete the table

negative vs positive regulation

1. Negative regulation

• Repressor protein binds to DNA

• Prevents transcription

2. Positive regulation

• Activator protein binds to DNA

• Activates transcription

negative vs positive regulation of the lac operon

Lac operon (negative)

• Ligand binds to repressor protein

• Repressor protein removed

• Transcription activates

• Allolactose (ligand) is an inducer

Lac operon (positive)

• In the absence of glucose

• cAMP must bind to CAP (activator protein)

• cAMP is an inducer

• CAP-cAMP dimer binds to promoter

• Activates transcription

regulatory DNA binding proteins (list 3)

1. Trp repressor TrpR

• Binds to trp operon in presence of tryptophan

• Blocks transcription

 

1. Lac repressor LacI

• Binds to lac operon in absence of lactose

• Blocks transcription

 

1. CAP activator

• Binds to lac operon in absence of glucose

• Promotes transcription

repressors recognise (?) DNA sequence

operator

CAP recognises (?) binding site

CAP

in most cases, the protein inserts into the (?) of the DNA double helix

major groove

(?) or so bonds between the DNA binding protein and the DNA

20

helix-turn-helix motif

• common DNA-binding motif made up of two alpha-helices

• allows regulatory proteins to bind

DNA binding proteins bind as dimers

two copies in each major groove separated by exactly one turn of the DNA helix

lac repressor is a (?)

tetramer → two identical binding sites

how does the lac repressor prevent transcription of the lac operon

Lac repressor has 2 identical binding sites

Lac operator has three binding sites (O1, O2, O3)

Repressor binds O1 and either O2 or O3

Forms DNA loop

Loop contains the -35 and -10 binding sites (promoter regions) recognised by RNA polymerase

These sites are now inaccessible to RNA polymerase

where does CAP bind to in DNA?

CAP-binding site (major groove) adjacent to the promoter of the lac operon

upon binding, CAP-cAMP bends the DNA by (?) degrees

90 → stimulates transcription of the operon

trp repressor action

Trp present (co-repressor)

Binds to repressor protein

Changes shape -> can bind tightly to operator sequence

Blocks transcription of the enzymes required to synthesise trp

summary