An example that has been studied in great depth concerns the enzyme β-galactosidase, used by E. coli to convert the disaccharide lactose into its sugars constituent:
Lactose −−−−−−−−→ Glucose + Galactose
- β-galactosidase has been more studied as part of the lac operon of E. coli.
- This is made up of three structural genes designated Z, Y and A, which are clustered together and share a common promoter and terminator.
- The genes code for lac operon, such as: β-galactosidase, a permease and a transacetylase.
- The permease is necessary for the transport of lactose into the cell, while the role of the transacetylase is not entirely clear, although it is essential for the metabolism of lactose.
- Grouping the three genes together in this way ensures an ‘all-or-nothing’ expression of the three proteins.
- Transcription of these structural genes into their respective mRNAs is initiated by the enzyme RNA polymerase binding to the promoter sequence.
- However, this is only possible in the presence of lactose; in its absence, a repressor protein binds to an operator site, adjacent to the promoter, preventing RNA polymerase binding to the promoter, and therefore preventing mRNA production.
- Production of the inhibitor protein is encoded by a regulator gene (I), situated slightly upstream from the promoter and operator.
How then, the presence of lactose overcome this regulatory mechanism?
- Allolactose, an isomer of lactose and an intermediate in its breakdown, attaches to a site on the lac repressor, thereby reducing the latter’s affinity for the operator, and neutralising its blocking effect.
- The structural genes are then transcribed into mRNA, that is afterward translated into the 3 proteins described above, and the lactose is broken down.
- In the absence of lactose, there are only trace amounts of β-galactosidase present in an E. coli cell; this increases some 1000-fold in its presence.
(a) In the absence of the substrate lactose, the lac operon is ‘switched off’, due to a repressor protein encoded by the regulatory gene I. The repressor binds to the operator site, preventing the binding of RNA polymerase to the
promoter and therefore blocking transcription.
(b) Allolactose acts as an inducer by binding the repressor protein and preventing it from blocking the promoter site. Transcription of the three structural genes is able to proceed unhindered
- When all the lactose has been used, the repressor protein is free to block the operator gene once more, and the synthesis of further β-galactosidase ceases.
- The lac operon can also be induced or evoked by isopropyl β-thiogalactoside (IPTG); Escherichia coli is not able to break this down, so the genes remain permanently switched on.
- IPTG is used as an inducer in cloning systems involving the expression of the lacZ gene on pUC plasmids.
- The lac operon is subject to control or manage by positive also as negative regulator proteins.
- Transcription of the operon only occurs if another regulatory protein called catabolite activator protein (CAP) is bound to the promoter sequence.
- This is dependent on a relatively high concentration of the nucleotide cAMP which only occurs when glucose is scarce.
- The activation of the lac operon occurs only if lactose is present and glucose is (almost) absent or finished.