Module 4 Section 6

Question 1

Regulation of gene expression

Answer 1

• need to balance speed vs. efficiency
• only certain proteins need to be expressed at certain times
• expressing all proteins at all times wastes energy, regulation of transcription saves energy

Question 2

Lac Operon

Answer 2

-bacteria only express the genes for metabolizing lactose when lactose is present and glucose is scarce b/c lactose takes more steps to metabolize
-LacZ: B-galactosidease
-LacY: galactoside permease
LacA: Thiogalactoside transacetylase
LacO: Operator, regulates transcription
LacI: encodes LacI repressor protein (interacts with LacO)

Question 3

Galactosidase permease

Answer 3

channel protein, allows lactose in

Question 4

B-galactosidase

Answer 4

-metabolizes lactose

Question 5

Thiogalactoside transacetylase

Answer 5

modifies toxic galactosides that are imported with lactose

Question 6

Polycistrinsic mRNA

Answer 6

• lac operon is polycistrinsic

- has 3 shine dalgarno sequences before each of the 3 protein ORF

Question 7

Studying regulatory regions of Lac Operon

Answer 7

• used merodipoid model (partailly diploid bacterium)
• lacI mutant with wild type (WT) lacI, WT lacI made up for the mutated lacI (lacI produces diffusible product)
• lacO mutant with WT lac O, WT could not rescue mutant lacO, products produced even when lacI present (lacO not diffusible)
• lac Z mutation on one operon, lac O, lac Y other, only lac Z and lac A produced proteins

Question 8

Lac repressor

Answer 8

• negative regulation
• the operator has 3 sequences, all with same sequence
• repressor binds to 2 of 3 operator sites (always O1), ties DNA in loop
• each site is inverted repeat
• binding protein is homotetrameric (2 dimers)
• O1 is just 3’ of promoter region
• O2 is in lac Z gene
• O3 is 5’ of the promoter

Question 9

Negative regulation

Answer 9

-binding of a repressor protein that prevents or decreases expression
-can act by:
blocking binding of RNA Pol to promoter
prevent closed to open conformational change
lock RNA Pol at the promoter

Question 10

If repression is absolute, how does Lac operon become activated

Answer 10

• repression is leaky
• off = transcription reduced 1000x, some copies still transcribed, allows galactoside permease to be produced, allows lactose into cell

Question 11

Lac operon activation

Answer 11

Allolactose: allosteric effector

• binds at a site other than DNA binding site
• changes conformation of the repressor, which weakens affinity for DNA
• Allolactose is special kind of allosteric effector known as an inducer

Question 12

Role of effectors in neg regulation

Answer 12

• regulate the binding of a repressor to DNA
• usually a small mlc or other protein that binds the repressor and causes a conformational change that results in an increase or decrease in transcription

Question 13

Positive regulation of Lac operon

Answer 13

• glucose is preferred energy source (for E.coli)
• metabolism of lactose in presence of glucose is blocked by catabolite repression (form of pos regulation)
• the activator is cAMP receptor protein

Question 14

cAMP receptor protein (CRP)

Answer 14

• Lac operon activator
• homodimer
• each subunit binds cAMP (cyclic adenosine monophosphate)
• cAMP is required for CRP to bind DNA
• cAMP IS THE EFFECTOR
• increase [glucose]=decreased cAMP production, increased cAMP export, decreased [cAMP]
• decreased [glucose] is vise versa

Question 15

Constitutive gene expression

Answer 15

• genes for products that are needed at all times

- AKA ‘housekeeping genes’

Question 16

Gene regulatory networks

Answer 16

-the nodes of the maps (where the lines intersect) are master regulators of the gene program

Question 17

DNA binding motifs

Answer 17

• helix-loop-helix motif commonly found in eukaryotic transcription factors
• proteins share a conserved region of ~50 residues
• contains two amphipathic a-helices linked by loop
• one helix mediates dimer formation
• DNA binding mediated by other helix (recognition helix), has basic residues to enable DNA binding
• these are on each monomer

Question 18

Coactivator/corepressor

Answer 18

• coactivators act as bridge between activator and RNA Pol to activate transcription
• corepressors act as a block to inhibit binding of the activator to the RNA Pol. and prevent transcription

Question 19

Helix-loop-helix heterodimers

Answer 19

• can form heterodimers composed fo similarly structured proteins
• creates larger number of functional transcription factors from a smaller number of individual proteins
• are less likely to bind inverted repeats than homodimers