How does allolactose induce transcription ofthe lac genes

Question

Biology Lac Operon Assignment

Question 1

a) How does allolactose induce transcription ofthe lac genes?

1) Lactose binds to the polymerase and increasesefficiency.

2) Lactose binds to a repressor protein, and alters its conformation to prevent it from binding to the DNA and interfering with the binding of RNApolymerase.

3) Lactose binds to an activator protein, which can then help the RNA polymerase bind to the promoter and begintranscription.

4) Lactose prevents premature termination of transcription by directly binding to and bending the DNA.

b) Which molecule signals to the Lac operon that glucose levels are low?

1) CyclicAMP
2) Calcium
3) Lactose
4) Pyruvate

Question 2

In lab, you are conducting experiments to study the lac operon in E. coli (see schematic below). In your experiments, the activity of the enzyme b-galactosidase (Π -gal) is measured by including X-gal and IPTG in the growth media. X-gal is a lactose analog that turns blue when metabolized by b-gal, but it does not induce the lac operon. IPTG is an inducer of the lac operon but is not metabolized by b-gal.

a) Which of the following would you expect to bind to Π-galactosidase? Circle all thatapply.

Lactose (or allolactose)             X-gal                IPTG

b) Which of the following would you expect to bind to the lac repressor? Circle all thatapply.

Lactose (or allolactose)             X-gal                IPTG

After mutagenesis you find 7 mutants that never turn blue as shown in the table below. *Note each mutant has a single loss-of-function mutation.

I: LacI gene
CAP binding site: also known as the CRP binding site
Plac: the promoter of the lac operon
O: operator (can be O₁, O₂, OR the O₃)

Cell Type	Media
	+ glucose No lactose + X-gal	+ glucose + lactose + X-gal	No glucose No lactose + X-gal	No glucose + lactose + X-gal
Wild type	White colonies	White colonies	White colonies	Dark blue colonies
Mutants 1-7	White colonies	White colonies	White colonies	White colonies

Wild type White colonies White colonies White colonies Dark blue colonies

Mutants 1-7 White colonies White colonies White colonies White colonies

c) A single loss-of-function mutation in which component or components (I,CAP binding site, Plac, O₁, O₂, O₃, lacZ) could produce the un-inducible phenotype seen in these mutants? List all thatapply.

You also find three mutants with the following phenotype. *Note that each mutant has a single loss-of-function mutation.

Cell Type	Media
	+ glucose No lactose +X-gal	+ glucose + lactose + X-gal	No glucose No lactose + X-gal	No glucose + lactose + X-gal
Wild type	White colonies	White colonies	White colonies	Dark blue colonies
Mutants 8-10	White colonies	White colonies	Dark blue colonies	Dark blue colonies

d) A loss-of-function mutation in which component or components (I, CAP binding site, Plac, O₁, O₂, O₃, lacZ) could produce the constitutive phenotype seen in thesemutants?

e) You introduce a piece of DNA into the constitutive mutant 8 bacterial cells that contains the following: lacI and its promoter, lacZ, lacA, and lacY and their promoter (Note: all of these genes are wild-type). (Introducing additional DNA is a common laboratory technique in bacteria, and all added genes and regulatory regions can be expected to act as if they were a part of the genome.) However, the addition of introducing your piece of DNA with wild-type lac genes does not rescue the mutant phenotype observed in mutant 8; that is, these bacteria are still constitutive. Does this additional information allow you to narrow your options as to possible causative mutations? Clue: What mutations would prevent the lac operon from being inducible (in other words, repressed)?