L3, L4, L5 - The Lac Operon Flashcards
What is the Lac Operon?
The LO is an operon required for the transport and metabolism of lactose in E.coli, and many other enteric bacteria.
Its function is to allow E.coli to grow on lactose
What is an Operon?
An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter.
The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product.
The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.
What 2 enzymes does E.coli need to metabolise lactose?
1) Lactose permease - transports lactose into the cell
2) B-galactosidase - hydrolyses lactose to glucose and galactose
What 3 genes does the Lac Operon contain? How are they transcribed?
3 genes that are transcribed together:
1) lacZ (z) - codes for B-galactosidase
2) lacY (y) - codes for lactose permease
3) lacA (a) - codes for B-galactosidase transacetylase
Under what 2 conditions is the LO transcribed?
E.coli uses glucose preferentially LO is only transcribed when its products are needed, which is:
When lactose is present AND glucose is absent
(around 5000 molecules of B-galactosidase per cell)
What kind of regulation molecule is seen with the LO? What are the mutants of this molecule?
1) increasing transcription when lactose is present (activator)
2) decreasing transcription when lactose is absent (repressor)
The regulator is a repressor protein.
This was proved because the mutants of it were constitutive. Gene mapping shows that these mutants are in a gene near but not part of the LO, so mutation didn’t directly effect the operon itself. This gene was named lacI (inducibility)
What does LacI code for and what does it do if:
a) lactose is absent from the growth medium
b) lactose is present and glucose is also absent
The lacI gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive).
a) If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the lac operator. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore mRNA encoding LacZ, and LacY is only made at very low levels.
b) When cells are grown in the presence of lactose however, a lactose metabolite called allolactose (made from lactose by the product of the lacZ gene) binds to the repressor, causing an allosteric shift. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.
What is the lacI gene?
A gene near to the LO that codes for the repressor of the LO
What is allolactose and what does it do when:
a) Lactose is absent
b) Lactose is present
Allolactose is the natural signal molecule, it is an isomer of lactose. SO therefore is present when lactose is.
Allolactose is produced from lactose by the small amount of B-galactosidase present before induction.
1) Lactose absent: repressor protein binds to the operator blocking transcription
2) Lactose present: allolactose binds to the repressor and causes it to dissociate from the operator, allowing transcription
How do you find out where RNAP binds to the DNA of the LO?
It is tested by doing ‘in vitro DNAase protection experiments’:
If no protein is bound to DNA, DNAase will break it down into small pieces.
But if a protein (such as RNAP) is bound to the DNA it protects it from DNAase
Sequencing the protected DNA chains shows where the protein is bound to it.
What do DNAase protection experiments show about the binding sites of the Lac Repressor and RNAP?
It shows that they have overlapping binding sites. 2 Lac repressor molecules bind to the operator to block the passage of RNAP
Describe the structure of the Lac Repressor
The lac repressor is a four-part protein, a tetramer, with identical subunits.
Each subunit contains a helix-turn-helix (HTH) motif capable of binding to DNA.
The operator site where repressor binds is a DNA sequence with inverted repeat symmetry therefore the 2 DNA half-sites of the operator together bind to 2of the subunits of the repressor.
The other 2 subunits of repressor are not doing anything in this model
AS well as the original operator (O1) there are 2 more operators (O2 and O3). Describe the significance of O2 and O3
Bind of the Lac repressor to O1 reduces transcription by x20.
But binding of the Lac Repressor to O1 and either O2 or O3 (not both) reduces transcription by x1000
What kind of binding is between O1 and either O2 or O3, and what does it do to the DNA between them?
Binding is co-operative and the DNA between the 2 bound operons probably forms a loop:
O1 + O2 = partly looped
O1 + O3 = promoter is a loop
What causes the repressor to dissociate in the presence of lactose?>
When lactose is present allolactose is too, and this binds to the Lac Repressor and coause a conformational change. The hinge region is disordered and subunits don’t bind co-operatively to the operator.
