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Genes, Genomes and Biomedical Implications > Lac Operon > Flashcards

Lac Operon Flashcards

1
Q

What is abortive initiation?

A

the repetitive synthesis and release of short nascent RNAs by RNAP

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2
Q

What is scrunching of DNA?

A

the change in conformation during initiation of transcription

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3
Q

What is ChIP-seq used for?

A

to study protein-DNA interactions on a genome-wide scale

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4
Q

What is a regulator gene?

A

a gene that encodes for a product that controls the expression of other genes

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5
Q

What is an operon?

A

a unit of bacterial gene expression and regulation, consisting of a promoter, operator and structural genes

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6
Q

What does a cis-acting site affect?

A

the activity only of sequences on its own molecule of DNA (or RNA) i.e. the site does not code for protein

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7
Q

What does a trans-acting product suggest?

A

it is a diffusible protein or RNA

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8
Q

What happens in negative control?

A

a repressor protein binds to an operator to prevent a gene from being expressed

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9
Q

What happens in positive control?

A

a TF (activator) is required to bind at the promoter in order to enable RNA polymerase to initiate transcription

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10
Q

What does LacI gene have?

A

its own promoter and terminator

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11
Q

What is transcription of the LacZYA operon controlled by?

A

the lac repressor that binds to an operator that overlaps the promoter at the start of the cluster (PO)

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12
Q

What is the polycistronic transcription unit of the LacZYA operon made up of?

A
  • promoter
  • lacZ, Y and A
  • terminator
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13
Q

What does β galactosidase do?

A

break down and hydrolyse lactose into glucose and galactose

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14
Q

What does permease do?

A

help transport the beta galactosidase into the cells

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15
Q

How big is the lac operon operator?

A

the first 26 bp of each transcription unit

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16
Q

Where does the lacZ gene start on the transcription unit?

A

base 39

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17
Q

What is the lac repressor?

A

a tetramer of identical subunits coded by the lacI gene

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18
Q

What is the lac repressor controlled by?

A

an inducer

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19
Q

What does the lac inducer do?

A

convert the lac repressor to a form with lower operator affinity to allow RNAP to initiate transcription

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20
Q

What are the 2 binding sites on the lac repressor?

A
  • one for the operator DNA
  • one for the inducer
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21
Q

What does lacZ encode for?

A

β galactosidase

22
Q

What is IPTG?

A

a gratuitous inducer i.e. a molecular mimic of allolactose

23
Q

What does IPTG do?

A

trigger transcription of the lac operon and induce protein expression where the gene is under the control of the lac operator

24
Q

What are the 3 components of a single repressor subunit?

A
  • N-terminal DNA-binding domain
  • a hinge
  • the core of the protein
25
Q

What does the DNA-binding domain of the repressor subunit contain?

A

two short α-helical regions (helix-turn-helix) that bind the major groove of DNA

26
Q

What do all HTH DNA binding proteins do?

A

bind DNA as dimers that are 3.4 nm apart

27
Q

What do the amino acid side chains of the C-terminal helix of the HTH do?

A

recognise the specific DNA sequence to which the protein binds

28
Q

What is the lac operator?

A

a palindromic sequence of 26 bp consisting of adjacent inverted repeats

29
Q

What is a palindrome?

A

a DNA sequence that reads the same on each strand of DNA when the strand is read in the 5’ to 3’ direction

30
Q

What does each inverted repeat of the lac operator do?

A

bind to the DNA-binding site of one repressor subunit

31
Q

What does inducer binding to the repressor do?

A

cause a change in repressor conformation that reduces its affinity for DNA and releases it from the operator

32
Q

Give examples of DNA motifs recognised by bacterial regulator proteins

A
  • lac repressor
  • catabolite activator protein (CAP)
  • lambda repressor
33
Q

What do mutations in the cis-dominant operator cause?

A

constitutive expression of all 3 lac structural genes because the operator is unable to be bound with repressor protein i.e. RNAP has unrestrained access to the promoter

34
Q

What can the cis-dominant operator control?

A

only adjacent lac genes

35
Q

What do mutations that inactivate the lacI gene cause?

A

the operon to be constitutively expressed because the mutant repressor protein cannot bind to the operator

36
Q

What is meant by the lacI- gene being repressive?

A

induction of a normal lacI+ gene can restore control even in the presence of a defective lacI- gene

37
Q

What do mutations in the inducer-binding site of the repressor do?

A

prevent lac operon transcription which causes uninducibility

38
Q

What explains the dominant negative behaviour of the lacI-d mutation?

A

mutant subunits can combine with normal subunits and interfere with their function; it only requires 1 subunit of the multimer to be lacI-d to block repressor function

39
Q

What does full repression of the lac operon require?

A

the repressor to bind to an additional operator downstream (O2) or upstream (O3), as well as to the primary operator at the lacZ promoter (O1)

40
Q

What is catabolite repression?

A

the ability of glucose to prevent the expression of a number of genes

41
Q

What does cAMP do?

A

convert inactive CAP to a form that binds the promoter and assists RNAP in initiating transcription

42
Q

What activates a dimer of CAP?

A

a single molecule of cAMP

43
Q

What does CAP do?

A

activate the α subunit of RNAP by interacting with the CTD

44
Q

What is cAMP controlled by?

A

the level of glucose in the cell; a low glucose level allows cAMP to be made

45
Q

What happens to the lac operon in the presence of both glucose and lactose?

A
  • β-galactosidase is not needed
  • lac operon is off
  • glucose causes low levels of cAMP, and CAP fails to bind to its site in the lac operon
46
Q

What happens to the lac operon in the presence of only glucose?

A
  • β-galactosidase is not needed
  • lac operon is off
  • lac repressor bind to its operator and CAP fails to bind
47
Q

What happens to the lac operon in the presence of only lactose?

A
  • β-galactosidase is needed
  • CAP binds and turns the lac operon on
48
Q

What happens to the lac operon in the absence of both glucose and lactose?

A
  • β-galactosidase is not needed
  • lac operon is off because Lac repressor bound to its operator prevents CAP from turning the lac operon on
49
Q

What are the 2 agar plates used to examine the lac+ phenotype?

A
  • one has nutrient agar containing IPTG and X-gal, which is cleaved by beta-galactosidase to produce a blue reaction product
  • the other is a control and contains nutrients and X-gal only
50
Q

What is blue-white screening used for?

A

the detection of successful ligation in vector-based gene cloning

Genes, Genomes and Biomedical Implications (15 decks)

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