You have systematically mutagenized the lac operon in E. coli to produce a mutat

Question

You have systematically mutagenized the lac operon in E. coli to produce a mutation that disrupts the function of each of the following elements:

a. the promoter for LacI (P(I))

b. the LacI gene

c. CRP binding site

d. the promoter for the lac operon (P(lac))

e. the operator sequence

f. a mutation in lacZ that disrupts the coding region but does not disrupt transcription

g. a mutation in lacZ that blocks transcription

For each of the above mutations, what would be the phenotype of the mutant cell growing in high lactose/low glucose conditions? High glucose/low lactose conditions? Explain your reasoning

Explanation / Answer

Functions of the genes concerned with lac operon are:

lacI- Gene for the repressor protein, it blocks the transcription of lacZYA in the absence of lactose.

lacO- codes for the operator protein which with the help of promoter helps the RNA polymerase to bind to the operon.

lacZ- codes for -galactosidase

lacY- codes for -galactoside permease

lacA- codes for -galactoside transacetylase

(a) When E. Coli strain is mutated with the promoter for LacI (P(I)) the lac operon would not function in either situation as the promoter region is responsible for beginning the transcription of the entire set of the structural gene for lactose digestion.

(b) Mutation in LacI will lead to the constitutive formation of LacZ gene because, LacI produces a protein that blocks the binding of the enzyme RNA polymerase to the promoter, in the absence of lactose. This blocking stops and the promoter gets activated when allolactose binds to the lacI protein. Hence, in such a situation, the presence of even a small amount of lactose or even in the absence of lactose the LacZYA genes are transcribed.

(c) Mutation in CRP binding site will make the binding of CRP (cAMP Receptor Protein) to cAMP. It has been seen in experiments and has been established that CRP can help in the binding of the promoter to RNA polymerase molecule. Any mutation in the binding site of this protein will lead to a decreased level of LacZYA expression in high lactose/low glucose environment and little or no expression in low lactose and high glucose environment.

(d)Any mutation in promoter gene will lead to the non-functionality of the lac operon.

(e)  Mutation in operation means in the loss of operator function, the presence of lacZ gene will lead to the constitutive formation of LacZ Protein as the repressor(lacI) cannot bind to the operator both in low /high lactose environment.

(f) a mutation in lacZ that disrupts the coding region but does not disrupt transcription will lead to the formation of -galactoside permease and -galactoside transacetylase but not -galactosidase. This will hamper the lactose digestion function of the lac operon as a whole, as all the enzymes are crucial for it functioning. but if -galactosidase is added from outside, the entire operon will run smoothly.

(g) a mutation in lacZ that blocks transcription entirely will render the operon useless as no transcription means no enzymes for lactose digestion.

In each scenario, it is mandatory to know that, lac operon will start functioning in the presence of even a little amount of lactose( other than the situations a,d and g).

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