Question

The lac operon contains a DNA sequence known as the lac promoter (P or P+ for wild type; P– for mutant (RNA polymerase does not bind)) that serves as the RNA polymerase binding site. The lac operon also contains a DNA sequence known as the Lac operator (O or O+ for wild type; O– or Oc for mutant (lac repressor cannot bind)) which is the binding site for lac repressor. The lac repressor, a protein, is encoded by the lac repressor gene known as lacI. The lacI gene has its own promoter and is not part of the lac operon. The lac operon also contains the structural genes coding for the three enzymes required for the import and breakdown of lactose. In the absence of lactose, the operator is occupied by the lac repressor, preventing the binding and functioning of RNA polymerase. When lactose is present in the cell, an lactose is enzymatically rearranged to form allolactose, the allolactose can bind to the lac repressor changing its conformation and reducing its affinity for the lac operator. In the presence of lactose, the lac repressor does not bind well to the lac operator. This is a negative control mechanism - the lac operon is on unless turned off by the binding of lac repressor to the operator. The lac operon can be induced to be on by the presence of lactose. IPTG is an artificial compound that will bind to the lac repressor and cause the same allosteric change in the lac repressor as does allolactose. The lac operon also has a positive control of expression. The catabolite activating protein or CAP, must be bound at the CAP binding site in order for transcription to proceed, even if the RNA polymerase is bound to the promoter. CAP requires the binding of cAMP in order to bind to the CAP binding site. In the presence of glucose, the enzyme adenylate cyclase is inactive, cAMP is not produced, and CAP is not able to bind upstream of the promoter (this is required for stable RNA polymerase binding). In the absence of glucose, the adenylate cyclase is active, cAMP levels increase and when cAMP binds to CAP, CAP binding to the CAP binding site increases. When CAP is bound, it interacts with the adjacent RNA polymerase increasing the rate at which the RNA polymerase is able to leave the promoter and continue transcription. This dual regulation ensures that the lactose operon is expressed at a high level only in the presence of lactose and when glucose, a preferred energy source, is absent. The operon consists of the promoter that serves as the RNA polymerase binding site, the operator, attachment site for lac repressor, and the structural genes coding for the three enzymes required for the breakdown of lactose. In the absence of lactose, the operator is occupied by the lac repressor, preventing the binding and functioning of RNA polymerase. The other “branch” of regulation is controlled by glucose in the medium. In the presence of glucose adenylate cyclase is inactive, cAMP is not produced, and CAP is not able to bind upstream of the promoter (this is required for stable RNA polymerase binding). In the absence of glucose, the adenylate cyclase/cAMP/CAP axis is activated. This dual regulation ensures that the lactose operon is expressed at a high level only in the presence of lactose.

THE EXPERIMENT Two mutant E. coli strains were compared in this study. In one strain, the gene coding for adenylate cyclase has been mutated and the enzyme is inactive. In the other mutant strain of E. coli, the lacI gene has been mutated and the lac repressor DNA binding domain has been eliminated.

For the following questions/statements, These are the following responses:

A. if the statement is associated with adenylate cyclase mutant cells only;

B. if the statement is associated with lac repressor mutant cells only;

C. if the statement is associated with both mutants;

D. if the statement is associated with neither mutant.

1. These cells would become normal if transformed by an expression plasmid containing lacI, the lac repressor gene.

2. These cells would become normal if transformed by a plasmid containing the lac operator.

3. If present, the lac repressor would be unable to bind to the operator under any conditions in these cells

Answer 1

1) the cells lacking lac I when plasmid containing lacI gene is given to the cell functional lac repressor can be produced which can bind to the operator of the lac operon present in the bacterial chromosome so cells lacking functional lacI gene become normal if transformed by an expression plasmid containing lacI, they lac repressor gene so the answer is B. if the statement is associated with lac repressor mutant cells only

2) cells lacking functional lac repressor and adenylyl cyclase both have functional lac operator so there will be no change when cells are transformed with a plasmid containing lac operator so D. if the statement is associated with neither mutant.

3) lac repressor can bind to the lac operon with functional lac operator here both mutant cells have functional lac operator so D. if the statement is associated with neither mutant.