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LSM2232 > DLW02 - The Lac Operon > Flashcards

DLW02 - The Lac Operon Flashcards

1
Q

Define “regulator gene”

A

A regulator gene is a gene that codes for a product that controls the expression of other genes.

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2
Q

Define “operon”

A

An operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognised by regulatory gene products.

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3
Q

Describe negative control

A

In negative control, a repressor protein binds to an operator to prevent a gene from being expressed.

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4
Q

Describe positive control

A

In positive control, an activator (transcription factor) binds to the promoter to enable RNA polymerase to initiate transcription

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5
Q

How is induction achieved?

A

Induction can be achieved by activating an activator or inactivating a repressor.

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6
Q

How is repression achieved?

A

Repression can be achieved by activating a repressor or inactivating an activator

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7
Q

What is meant by the term “negative inducible”?

A

Negative - a repressor protein is involved.

Inducible - to activate the gene, the repressor protein must be inactivated.

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8
Q

The lacI gene shares the same promoter as the lac structural genes. TRUE or FALSE?

A

FALSE. The lacI gene has its own promoter and terminator.

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9
Q

State the product of the gene lacZ and its function

A

beta-galactosidase: metabolises complex beta-galactoside sugars such as lactose

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10
Q

State the product of the gene lacY and its function

A

permease: transports beta galactoside into cells

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11
Q

State the product of gene lacA and its function

A

transacetylase: transfers an acetyl group from acetyl-CoA to beta-galactoside

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12
Q

State the inducer of the lac operon

A

allolactose, the isomer of lactose

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13
Q

State the substrate of the lac operon

A

beta galactoside

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14
Q

beta-galactosidase (the protein) degrades at a similar rate as the mRNA encoding it. TRUE or FALSE?

A

FALSE. The lac mRNA is extremely unstable, and degrades before beta-galactosidase.

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15
Q

Define “gratuitous inducer”

A

A gratuitous inducer is an inducer that resembles authentic inducers of transcription but are not substrates for the induced enzymes. Hence, they cannot be metabolised and persist in the cell.

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16
Q

The lac repressor is a multimeric enzyme. How many subunits does it contain?

A

Four.

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17
Q

Briefly describe the formation of a tetrameric lac repressor.

A

The lac repressor is synthesised as a monomer. Two monomers form a dimer by making contacts between core 1 and 2.
Two dimers then form a tetramer by interactions between the tetramerisation (oligomerisation) helices.

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18
Q

Describe the structure of a monomer of the lac repressor

A

The monomer contains:

i) a helix-turn-helix DNA binding domain,
ii) two core subdomains
iii) an oligomerisation helix
iv) a hinge
v) an inducer binding site.

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19
Q

What is a helix-turn-helix?

A

A helix-turn-helix motif is one of the most common DNA binding motifs. All HTH DNA binding proteins bind DNA as dimers, in which the two copies are separated by exactly 3.4 nm (distance of the major groove)

20
Q

Suggest 3 ways that may cause constitutive expression of the lac operon and explain why.

A
  1. Mutation to lac operator region which disallows lacR to bind and thus no repression of lac operator.
  2. Mutation to DNA-binding domain of lacR prevents it from binding to lac operator
  3. Mutation to CRP that allows it to become constitutively active without cAMP
21
Q

What are cis-acting mutations?

A

Cis-acting mutations are mutations that affect only genes related to it on a contiguous stretch of DNA.

22
Q

What are dominant mutations?

A

Dominance can be defined as the inability to restore the wild-type phenotype (of the mutated gene) if a wild-type copy is present.

23
Q

What are trans-acting mutations?

A

Trans-acting mutations are mutations of the regulatory gene (i.e. trans-acting factors)

24
Q

Give three examples of trans-acting mutations of the lac operon and describe them briefly

A
  1. lacI^(-): a recessive mutation in the lac repressor, causing it to be inactive.
  2. lacI^(-d): a dominant mutation in the lac repressor, preventing it from binding to the operator.
  3. lacI^(s): a mutation in the lac repressor, preventing it from binding to the inducer.
25
Q

Why is the lacI^(-d) mutation dominant?

A

Even if a wild-type copy is present, mutant subunits synthesised from the mutated lacI can combine with normal subunits and interfere with their function. One mutant subunit is enough to block repressor function.

26
Q

Describe the lacI^(s) mutation.

A

This mutation causes the lac repressor to be unable to bind to the inducer. This results in the inability to release the lac repressor from the operator (super-supressor).

27
Q

How many operator regions are present in the lac operon?

A

Three. O1, O2 and O3.

28
Q

Briefly describe the full repression of the lac operon, taking into account the fact that the lac operon has multiple operators.

A

The lac repressor (lacR) initially binds to O1, as it has the highest affinity for O1. The same lac repressor then binds either O2 or O3, resulting in the bending of DNA. This causes full repression.

29
Q

The expression of the lac operon is controlled by glucose. TRUE or FALSE?

A

TRUE. Glucose levels are used as a second layer of control, involving catabolite repression.

30
Q

Describe the effect of glucose on lac operon expression

A

A lack of glucose allows cAMP to be made.
cAMP activates CRP (cAMP receptor protein)
CRP binds to lacC and interacts with the C-terminal domain (CTD) of the alpha subunit of RNA polymerase, activating it.

31
Q

Briefly explain the results one would expect in a blue-white screening experiment.

A

There are two possibilities: either a white colony is formed or a blue colony is formed. White colonies signify an absence is beta-galactosidase while blue colonies signify an active beta-galactosidase.

32
Q

What happens if X-gal is not added in a blue-white screening experiment?

A

All colonies would be white. X-gal is cleaved to form an insoluble blue pigment.

33
Q

Briefly describe the function of blue-white screening in determining successful ligation reactions in gene cloning.

A

Successful insertion of insert DNA into the plasmid vector disrupts the lacZ gene, resulting in the inability to express the lac phenotype.
Blue colonies indicate an unsuccessful ligation, while white colonies indicate some form of disruption at the MCS (multiple cloning site) containing the lacZ gene.

34
Q

IPTG plays an important role as an inducer in laboratory settings. State two ways IPTG is used to induce protein expression.

A
  1. blue-white screening

2. pET expression system for toxic genes.

35
Q

What is alpha-complementation?

A

Another name for blue-white screening.

36
Q

State the expression of the lac operon (ON or OFF) in the presence of both glucose and lactose

A

OFF.

37
Q

State the expression of the lac operon (ON or OFF) with glucose as the sole carbon source.

A

OFF.

38
Q

State the expression of the lac operon (ON or OFF) with lactose as the sole carbon source.

A

ON.

39
Q

State the expression of the lac operon (ON or OFF) in the absence of both glucose and lactose

A

OFF.

40
Q

State the three regulatory DNA sequences involved in the lac operon

A
  1. lacC, CAP/CRP binding site
  2. lacO, the lac operator: lac repressor binding site
  3. lacP, the lac promoter.
41
Q

Suggest the effect of the mutation CAP^(-)

A

The mutation results in an inactive CAP (cAMP receptor protein). This results in the inability of CAP to activate RNA polymerase, regardless of glucose levels. The structural genes are not synthesised.

42
Q

Suggest the effect of the mutation CAP^(c)

A

The mutation results cAMP-independent CAP. As such,m RNA polymerase is activated regardless of glucose levels, and structural genes can be synthesised in as long as lactose is present. As the name implies, this mutation is constitutive.

43
Q

Suggest the effect of the mutation lacP^(-)

A

The mutation results in an inactive promoter. Structural genes cannot be synthesised.

44
Q

Suggest the effect of the mutation lacC^(-)

A

The mutation results in CAP being unable to bind to lacC. RNA polymerase cannot be activated. Structural genes are not synthesised.

45
Q

Suggest the effect of the mutation lacO^(c)

A

The operator is mutated and cannot bind the repressor protein. Structural genes are constitutively expressed.

46
Q

Suggest the effect of the mutation lacZ^(-)

A

The gene encoding beta-galactosidase is inactive. Other structural genes are expressed.

LSM2232 (9 decks)

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