Regulation of gene expression
- 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
Lac Operon
-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)
Galactosidase permease
channel protein, allows lactose in
B-galactosidase
-metabolizes lactose
Thiogalactoside transacetylase
modifies toxic galactosides that are imported with lactose
Polycistrinsic mRNA
- lac operon is polycistrinsic
- has 3 shine dalgarno sequences before each of the 3 protein ORF
Studying regulatory regions of Lac Operon
- 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
Lac repressor
- 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
Negative regulation
-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
If repression is absolute, how does Lac operon become activated
- repression is leaky
- off = transcription reduced 1000x, some copies still transcribed, allows galactoside permease to be produced, allows lactose into cell
Lac operon activation
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
Role of effectors in neg regulation
- 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
Positive regulation of Lac operon
- 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
cAMP receptor protein (CRP)
- 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
Constitutive gene expression
- genes for products that are needed at all times
- AKA ‘housekeeping genes’
Gene regulatory networks
-the nodes of the maps (where the lines intersect) are master regulators of the gene program
DNA binding motifs
- 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
Coactivator/corepressor
- 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
Helix-loop-helix heterodimers
- 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
