Regulation of gene expression - mechanisms

Question 1

Structure of lac Operon

Answer 1

In E.coli
lac operon encodes 3 structural genes associated with lactose metabolism
- lacZ is beta-galactosidase, mutations in this does inhibit metabolism lactose
- lacY is lactose permease, mutations in gene does inhibit metabolism lactose
- lacA is galactosidase acetyltransferase, mutations in gene don’t inhibit metabolism of lactose
lac operon expressed only when cells need metabolise lactose
Expression lac operon controlled by regulatory gene lacl is negative regulator
lacl gene encodes lac repressor, binds lacO operator sequences and blocks RNAP activity

Question 2

lac Operon inducer - allolactose

Answer 2

Lactose is disaccharide of glucose and galactose
lacZ gene produces beta-gelactosidase hydrolyses lactose
Galactose phosphorylated and converted to glucose phosphate
Expression lac operon induced in presence lactose
Inducer molecule is lactose isomer, allolactose, generated in side reaction of beta-galactosidase
Allolactose binds repressor and stops its ability to repress

Question 3

lacl repressor binds both DNA and inducer

Answer 3

lacl repressor is homotetramer - 4 identical subunits (proteins) come together to make it
Protein has 3 domains:
- Tetramerization domain
- Core domain - binds inducer
- Head domain - binds operator sequence
When bound to inducer, repressor alters shape and can’t bind DNA
They bind inducers and DNA in mutually exclusive manner
Is an example of allosteric interactions
Repressor expressed constitutively (Lac expression inhibited in absence lactose)

Question 4

Binding lacl repressor causes DNA to twist

Answer 4

When 1 lacl repressor bind to 2 operator sequences, DNA binding sites each subunit align, causes DNA to twist and translation is unable to occur
Binding of lacl doesn’t prevent RNAP binding to P,lac promotor, but bound RNAP is transcriptionally inactive
RNAp primed to transcribe lac mRNA immediately upon binding inducer to repressor

Question 5

lac operon regulated by catabolite repression

Answer 5

Glucose preferred carbon sorce
In presence glucose genes required for metabolism other sugars is repressed
When glucose and lactose both present, lactose only metabolised after glucose exhausted
This causes diauxic growth curve
Glucose indirectly represses expression lac operon
Is called catabolite repression whenever glucose causes repression/down regulation enzymes
Expression lac operon requires both presence lactose and absence glucose

Question 6

Catabolite repression of lac operon blocks activation by cAMP and CAP

Answer 6

Mutations in genes encoding adenylate cyclase or catabolite activator protein (CAP) block expression lac operon
Adenylate cyclase makes signalling molecule cyclic AMP and is inhibited in presence of glucose
cAMP bind to CAP
cAMP/CAP complex bind to CAP site and required for DNAP activity and stimulates transcription
Only happens in absence glucose and presence lactose, both positively and negatively controlled

Question 7

elF2 critical for translation initiation

Answer 7

elF2 ind to charged initiator tRNA Met-tRNA and brings to the small ribosomal subunit
Once initiation codon localised elF2 hydrolyses GTP to GDP
Causes conformational change in elF2, releasing elF2/GDP from initiator tRNA

Question 8

elF2 is GTPase molecular switch

Answer 8

GTPase molecule switches such as elF2 undergo change in structural conformation upon GTP hydrolysis
Intrinsic GTPase activity of molecular switches is very low
GTPase activity stimulated by GTPase activating proteins (GAPs)
Each GAP specific to each GTPase
Recycling of GTPase required a guanine exchange factor (GEF), promotes release GDP
GTPase then recharge with GTP

Question 9

elF2 control point from translation control

Answer 9

elF2 activity can be rate-limiting for protein synthesis
Regulation pool of active elF2 can regulate amount protein synthesis in cell
Cells downregulate global translation in response various stress signals by depleting active pool of elF2
Possible stresses are ER stress, nutrient deprivation, viral infection and iron deprivation
This known as “integrated stress response” in mammalian cells
Protein kinases phosphorylate elF2 in response stress signals

Question 10

Phosphorylation elF2 affects how it bind elF2B

Answer 10

Phosphorylation elF2 doesn’t affect GTP hydrolysis, interaction with GEF elF2B or GDP release
But elF2-Pi binds tightly to elF2B
Is about 10 times more elF2 than elF2B in cell
Phosphorylated elF2 therefore rapidly depletes elF2B levels
Phosphorylation elF2 converts it from substrate of elF2B to inhibitor