Question

1. The role of the repressor gene was initially described with the aid of both single mutants and specifically constructed partially diploid strains of E. coli.

a.  List the phenotype ( Inducible, Constitutive, or Non-inducible ) of each of the following partial diploids with respect to the synthesis of beta-galactosidase and lactose permease. No explanation is necessary.

1)   i ^s p ⁺ o ⁺ z ⁺ y ⁺     // i ⁺ p ⁺ o ⁺ z ^- y ^-    

2)   i ^s p ⁺ o ⁺ z ^- y ^-       //     i ⁺ p ⁺ o ⁺ z ⁺ y ⁺

3)   i ⁺ p ⁺ o ⁺ z ⁺ y ⁺    // i ^-   p ⁺ o ⁺ z ^- y ^-   

4)    i ⁺ p ⁺ o ⁺ z ^- y ^-      // i ^- p ⁺ o ⁺ z ⁺ y ⁺  

b.  List the phenotype ( Inducible, Constitutive, or Non-inducible ) of each of the following double mutants with respect to the synthesis of beta-galactosidase and lactose permease. Explain your choice!

5)    i ^- p ^- o ⁺ z ⁺ y ⁺                

6)    i ^s p ⁺ o ^c z ⁺ y ⁺     

Answer 1

The lac operon is a mode of gene regulation in bacteria and involved in lactose metabolism. The operon is active in the presence of lactose. The operon is consists of the repressor (i), promoter (p), the operator (o), and structural genes sequences. When lactose is not present repressor protein bound to the operator region and inhibits the transcription. In the presence of lactose, repressor protein is not able to bind operator and binding of activator protein (CAP) to its binding site on promoter promotes the binding of RNA polymerase to the promoter and transcription of genes occur.

1) is p+ o+ Z+ y+ // i+ p+ o+ z- y- Non-inducible

Partial diploid has one active copy of genes beta-galactosidase and lactose permease (z+ and y-) and one non-functional/inactive copy of the gene (z- andy-). Both copies of the promoter and operator are functional (p+ and o+). This partial diploid is non-inducible because the super repressor (is) is always bound to the available operator site whether lactose is present or not. Mutation in gene sequence causes conformational changes in the repressor protein structure that affect the binding of inducer (lactose) to the repressor protein or make it always binds to the operator site. The repressor is a trans-acting protein so the super repressor binds to all operator site and make the operon non-inducible and inhibits the synthesis of beta-galactosidase and lactose permease.

2) is p+ o+ z- y- // i+ p+ o+ z+ y+  Non-inducible

This partial diploid will be non-inducible because of the presence of the super repressor.

3) i+ p+ o+ z+ y+ // i- p+ o+ z- y- Inducible

The lac operon is inducible in this partial diploid and will be active in the presence of lactose. It has one copy of normal active repressor protein which is bound to the operator site when lactose is not present. When lactose is present, it will bind to the repressor protein and inhibits its binding to the operator site and synthesis of beta-galactosidase and lactose permease will take place as the diploid has one functional copy of genes (z+ and y+).

4) i+ p+ o+ z- y- // i- p+ o+ z+ y+ Inducible

The lac operon is inducible and beta-galactosidase and lactose permease will be synthesized in the presence of lactose as the diploid has one copy of functional genes (z+ and y+).

5) i- p- o+ z+ y+ Non-inducible

This genotype has mutations in the promoter and repressor gene. The promoter is required for the transcription of genes and mutation in the promoter sequence inhibits the binding of RNA polymerase as a result transcription will not occur and beta-galactosidase and lactose permease will not be synthesized. Although due to mutation repressor protein will not bind to the operator site, a mutation in promoter makes lac operon non-inducible.

6) is p+ oc z+ y+ Constitutive

This genotype has a mutation in the repressor and operator region. The mutation in the operator region inhibits the binding of repressor protein and results in a constitutive operator (oc) which is always free for RNA polymerase binding irrespective of the presence of lactose in the medium. Super repressor can not bind to the constitutive operator (oc) and as a result lac operon is always active (constitutive) whether lactose is present or absent. So, beta-galactosidase and lactose permease will be synthesized constitutively.