MIC102 S11 - Coordination of Cell processes, Lac operon

Describe the different methods of regulating protein activity (competitive, non-competitive, adn covalent modifications)

Non-covalent

• Competitive

  • a molecule competes with the substrate for the active site of an enzyme → is reversible

  • blocks substrate binding without altering enzyme shape

  • can be overcome by increasing substrate concentration

• Non competitive (Allosteric)

  • Inhibitor binds to a site other than the active site (allosteric site) → is reversible

  • Change the enzyme’s shape, can no longer bind substrate

  • is NOT overcome by substrate concentration and can inhibit or activate enzyme fxn

Covalent

• Chemical Group covalently added or removed, altering structure and function

• ex: Phosphorylation, Methylation, Acetylation

Describe regulation of flux in central metabolism

• central metabolism is reversible to support catabolic and anabolic needs

• Cells regulate flux (flow through pathway) in response to:

  • level of energy

  • Concentration of reductant, precursors, and building blocks

• Activity of key enzymes in the pathway is regulated by allosteric activation or inhibition

What are the products of fueling in catabolic reactions of Central Metabolism?

1) ATP (Glycolysis and TCA)

2) Reducing power → NAD(P)H (PPP, Glycolysis, Transition Rxn, TCA) FADH₂ (TCA)

3) Precursor Metabolites

• ex: Pyruvate, Acetyl-CoA, Oxaloacetate, alpha-ketoglutarate, Ribose-5-phosphate

4) CO2 → Waste product (Transition Rxn, TCA)

What are different ways protein concentration can be regulated?

1) DNA topology

2) Promoter recognition

3) Trxn repression/activation

4) Trxn enhancement

5) Trxan termination

6)Regulatory sRNA

7) Messenger stability

8 ) Trln control

9) Proteolysis

Regulation of Flux - Glycolysis/GNG x PPP

Step 3: F6P → F1,6BP

• Forward Rxn

  • high concentration PEP (from PPP) → inhibits

  • high concentration of ADP → activates

• Reverse Rxn (GNG)

  • high concentration AMP → inhibits

Step 10: PEP → Pyruvate

• high concentration PEP → activates

Regulation of Flux - Transition Rxn

Pyruvate → Acetyl CoA

• Activators

  • PEP

  • AMP

• Inhibitors

  • Acetyl CoA

  • NADH

PEP → OAA

• Acetyl CoA → activates

• Aspartate → inhibits

Regulation of Flux - TCA

OAA → Citrate

• Inhibitory

  • NADH

  • alpha-ketoglutarate

General trend of reaction based on substrate and product concentration

High substrate concentration (need evergy for fueling)

• accelerate forward reaction

• ex: ADP/AMP

High product concentration (have energy for biosynthesis)

• slow forward reaction

• ex: ATP

No need to create excess energy if already sufficient → wasteful

Describe feedback inhibition

• the end-product inhibits an early enzyme in the pathway

• ex; histidine is an allosteric inhibitor of the first enzyme in its own biosynthetic pathway

What is the cAMP receptor protein and what does it do? When does it activate?

cAMP Receptor Protein (CRP)

• Catabolite Activator protein

• binds with cAMP to form CRP-cAMP complex

• binds to a CRP-cAMP binding site to activate trxn

cAMP - Cyclic AMP

• Only present when glucose is NOT being transported into cell

• without cAMP, transcription NOT activated

Therefore, expression of lac and other catabolite-utilization operons Only activated if glucose depleted

What is the LacI protein?

LacI

• protein in the lac operon, binds to operator region

• functions as a repressor → represses/inhibits trxn of lac((ZYA that metabolise lactose) genes when lactose NOT present

• If lactose present:

  • Lactose -(beta-galactosidase)→ allolactose (inducer)

  • binds to LacI → conformational change → can no longer bind to operator

  • repression of trxn is lifted

Lac Operon; Glucose +, Lactose -

CRP-cAMP bound

• No → no cAMP since glucose present

LacI bound

• Yes → no lactose, therefore no allolactose

Net effect on transcription

• not activated → no CRP-cAMP

• repressed → LacI bound

Relative level of lac mRNA expression

• Very low

Lac Operon; Glucose -, Lactose +

CRP-cAMP bound

• Yes→ cAMP present since no glucose

LacI bound

• No → lactose presnet so allolactose present

Net effect on transcription

• activated → CRP-cAMP bound

• derepressed → LacI NOT nound

Relative level of lac mRNA expression

• Maximal

Lac Operon; Glucose -, Lactose -

CRP-cAMP bound

• Yes→ cAMP present since no glucose

LacI bound

• Yes → no lactose, therefore no allolactose

Net effect on transcription

• activated → CRP-cAMP bound

• repressed → LacI bound

Relative level of lac mRNA expression

• Low

Lac Operon; Glucose +, Lactose +

CRP-cAMP bound

• No → no cAMP since glucose present

LacI bound

• No → lactose presnet so allolactose present

Net effect on transcription

• not activated → CRP-cAMP bound

• derepressed → LacI NOT bound

Relative level of lac mRNA expression

• Low

The lacI* strain carries a missense mutation that causes the LacI* protein to operate as if it is always bound to allolactose

Under which conditions will the lac opern be expressed at highest level in the lacI* mutant?

• presence of lactose and absence of glucose

• absence of lactose and absence of glucose

LacI protein acts ALWAYS BOUND to allolactose → therefore presnece of lactose DOESN’T matter