Question

Explain how elements of the lac operon are exploited to regulate recombinant protein production in E. coli.
this question is biochemistry question

Answer 1

The lac operon of E. coli contains genes involved in lactose metabolism. This is expressed only when lactose is present and glucose is absent.Two regulators turn the operon "on" and "off" in response to lactose and glucose levels -:  the lac repressor and catabolite activator protein (CAP).

The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present.The lac repressor senses lactose indirectly, through its isomer allolactose.Catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP

1. Structure of the lac operon

• The lac operon contains three genes: lacZ, lacY, and lacA.

1. The lacZ gene encodes an enzyme called β-galactosidase, which is responsible for splitting lactose (a disaccharide) into readily usable glucose and galactose (monosaccharides).

2. The lacY gene encodes a membrane protein called lactose permease, which is a transmembrane "pump" that allows the cell to import lactose.

3. The lacA gene encodes an enzyme known as a transacetylase that attaches a particular chemical group to target molecules. It's not clear if this enzyme actually plays any role in lactose breakdown

• The promoter is the binding site for RNA polymerase, the enzyme that performs transcription.

• The operator is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lacrepressor is bound, RNA polymerase cannot bind to the promoter and start transcription.

• The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.

2.The lac repressor

The lac repressor is a protein that inhibits transcription of the lacoperon. It does this by binding to the operator, which partially overlaps with the promoter.When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA.

This change in the lac repressor is caused by the small molecule allolactose,it is an example of an inducer.

Catabolite activator protein (CAP)

RNA polymerase alone does not bind very well to the lac operon promoter. It might make a few transcripts, but it won't do much more unless it gets extra help from catabolite activator protein (CAP). CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription.CAP isn't always active (able to bind DNA). Instead, it's regulated by a small molecule called cyclic AMP (cAMP). cAMP is a "hunger signal" made by E. coliwhen glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive.CAP is only active when glucose levels are low (cAMP levels are high). Thus, the lac operon can only be transcribed at high levels when glucose is absent.

The lac operon will be expressed at high levels if two conditions are met:

• Glucose must be unavailable: When glucose is unavailable, cAMP binds to CAP, making CAP able to bind DNA. Bound CAP helps RNA polymerase attach to the lac operon promoter.

• Lactose must be available: If lactose is available, the lac repressor will be released from the operator (by binding of allolactose). This allows RNA polymerase to move forward on the DNA and transcribe the operon.