The%20lac%20operon

1
The lac operon

• An example of positive and negative regulation of
  gene expression

2
Gene regulation

• Not every gene is transcribed in every cell, or
  at all times
• ? regulate transcription of specific genes in
  time and space

3

• NEGATIVE CONTROL

I
OFF
4

• NEGATIVE CONTROL

I
OFF
5

• NEGATIVE CONTROL

OFF
ON
6

• NEGATIVE CONTROL

I
OFF
I
gene
ON

• Release the brakes

7

• NEGATIVE REGULATION

I
OFF
I
gene
ON

• Release the brakes

8

• NEGATIVE REGULATION

I
OFF
I
ON

• Release the brakes

9

• NEGATIVE CONTROL

• POSITIVE CONTROL

I
OFF
I
ON

• Release the brakes

10

• NEGATIVE CONTROL

• POSITIVE CONTROL

I
OFF
I
ON

• Release the brakes

11

• NEGATIVE CONTROL

• POSITIVE CONTROL

I
OFF
I
ON

• Release the brakes

12

• NEGATIVE CONTROL

• POSITIVE CONTROL

I
OFF
I
ON
gene

• Release the brakes

13

• NEGATIVE CONTROL

• POSITIVE CONTROL

I
OFF
I
ON

• Press the accelerator

• Release the brakes

14
A classic example the bacterial lac operon

• genes in the lac operon encode proteins required
  to metabolise lactose
• LacZ b-galactosidase catalyses
• Lactose ? galactose glucose
• LacY Lac permease
• Transports lactose across cell membrane

15
Why regulate?

• Bacteria can use lactose

lactose
ON
The lac operon
16
Why regulate?

• Bacteria can use lactose
• BUT
• Its wasteful to make enzymes to metabolise
  lactose
• IF theres no lactose

17
Why regulate?

• Bacteria can use lactose
• BUT
• Its wasteful to make enzymes to metabolise
  lactose
• IF theres no lactose
• or
• IF glucose is available

18
What is an operon?

• Operon 2 genes with a single promoter

b
c
a
promoter
19
What is an operon?

• Operon 2 genes with a single promoter
• Genes transcribed as a single mRNA

AAAAA
20
What is an operon?

• Operon 2 genes with a single promoter
• Genes transcribed as a single mRNA
• This mRNA is POLYCISTRONIC
• encodes more than gene

21
What is an operon?

• Operon gt1genes with a single promoter
• Genes transcribed as a single mRNA
• This mRNA is POLYCISTRONIC
• encodes more than gene
• Coordinated production of proteins

Polycistronic mRNA
22
Structure of the lac operon
23
Structure of the lac operon

• 3 lac genes, lacZ, lacY and lacA

24
Structure of the lac operon

• 3 lac genes, lacZ, lacY and lacA

lacY
lacA
lacZ
b-galactosidase
PROTEINS
Lac permease
25
Structure of the lac operon

• 3 lac genes, lacZ, lacY and lacA
• All under the control of a single promoter

lacY
lacA
lacZ
promoter
b-galactosidase
Lac permease
26
Structure of the lac operon

• 3 lac genes, lacZ, lacY and lacA
• All under the control of a single promoter, which
  contains the operator site

lacY
lacA
lacZ
O
operator
promoter
b-galactosidase
Lac permease
27
Structure of the lac operon

• 3 lac genes, lacZ, lacY and lacA
• All under the control of a single promoter, which
  contains the operator site
• CAP site upstream

lacY
lacA
lacZ
O
operator
CAP site
promoter
b-galactosidase
Lac permease
28
Why regulate?

• The cell can use lactose
• as its sole carbon source
• BUT
• Theres no point making enzymes
• to metabolise lactose if theres no lactose

29
How does the cell turn these genes on or off
depending on lactose?
30
Without lactose
Lac Z
repressor
LacZ
LacY
LacA
O
31
Without lactose

• Lac repressor protein is bound to the operator

Lac Z
repressor
LacZ
LacY
LacA
O
32

• RNA polymerase cannot move past the promoter

Lac Z
RNA pol
LacZ
LacY
LacA
O
33

• RNA polymerase cannot move past the promoter

Lac Z
RNA pol
LacZ
LacY
LacA
O
34

• RNA polymerase cannot move past the promoter

• RNA polymerase cannot move past the promoter
• Transcription of lac genes is OFF

Lac Z
RNA pol
LacZ
LacY
LacA
O
35

• RNA polymerase cannot bind to the promoter
• Transcription of lac genes is OFF

Lac Z
36
The Lac repressor

• Lac repressor is encoded by the lacI gene
• lacI is upstream from the other lac genes
• lacI has its own promoter
• The repressor is constitutively expressed
• Its always available!

37
The Lac repressor

• Sequence-specific DNA-binding protein
• 4 subunits
• RNA polymerase cannot proceed if repressor is
  bound

38
When the cell needs to use lactose
The lac operon
39
When the cell needs to use lactose the lac genes
must turn ON
The lac operon
lactose
40

• How does the cell release the repressor ?

LacY
LacA
LacZ
O
LacZ
41

• When lactose is present

42

• When lactose is present
• Some lactose is converted to allolactose

43

• When lactose is present
• Some lactose is converted to allolactose

44

• When lactose is present
• Some lactose is converted to allolactose

45

• Allolactose binds to the repressor

46

• Allolactose binds to the repressor
• Binding changes the shape of the repressor

Wait for it!!
47

• Allolactose binds to the repressor
• Binding changes the shape of the repressor

48

• Allolactose binds to the repressor
• Binding alters the shape of the repressor
• The altered repressor cannot bind

49
LacZ
LacY
LacA
O
50
RNA pol
LacZ
51
RNA pol
LacY
LacA
LacZ
O
52
RNA pol
LacY
LacA
LacZ
O
53

• RNA polymerase can now move past the promoter

RNA pol
LacY
LacA
LacZ
O
54

• RNA polymerase can now move past the promoter
• Transcription is ON!

RNA pol
O
55

• RNA polymerase can now move past the promoter
• Transcription is ON!

RNA pol
O
56

• RNA polymerase can now move past the promoter
• Transcription is ON!

RNA pol
O
57

• RNA polymerase can now move past the promoter
• Transcription is ON!
• Enzymes are made

RNA pol
O
B-gal
Lac permease
58

• RNA polymerase can now move past the promoter
• Transcription is ON!
• Enzymes are made
• Lactose is metabolised!

lactose
RNA pol
O
59

• RNA polymerase can now move past the promoter
• Transcription is ON!
• Enzymes are made
• Lactose is metabolised!

lactose
glucose
galactose
60

• RNA polymerase can now move past the promoter
• Transcription is ON!
• Enzymes are made
• Lactose is metabolised!

lactose
glucose
galactose
61

• RNA polymerase can now move past the promoter
• Transcription is ON!
• Enzymes are made
• Lactose is metabolised!

lactose
glucose
galactose
62

• As lactose levels fall

RNA pol
O
63

• As lactose levels fall

RNA pol
O
64

• As lactose levels fall
• Allolactose releases the repressor

RNA pol
O
65

• As lactose levels fall
• Allolactose releases the repressor

• which changes shape

O
66

• As lactose levels fall
• Allolactose releases the repressor

• which changes shape

O
67

• As lactose levels fall
• ? Allolactose releases the repressor

• ? And can bind to the operator again

O
68

• As lactose levels fall
• ? Allolactose releases the repressor

• Prevents transcription

O
69

• As lactose levels fall
• Allolactose releases the repressor
• Allolactose is metabolised too

• Prevents transcription

O
70

• Prevents transcription

O
71
TO SUMMARISE
72
without lactose
Lac Z
x
Enzymes not needed
73
without lactose genes OFF
Lac Z
x
74
without lactose ?genes OFF
Lac Z
x
with lactose ? genes ON
75

• Lac genes are
• INDUCED
• by lactose

76
So far so good

• But the story is a little more complicated!

77
REMEMBER..

• The cell prefers to use glucose

78
REMEMBER..

• The cell prefers to use glucose
• WHY?

79
REMEMBER..

• The cell prefers to use glucose
• WHY?
• Glucose is nearly always available

80
REMEMBER..

• The cell prefers to use glucose
• WHY?
• Glucose is nearly always available
• Can be used without modification

81
REMEMBER..

• The cell prefers to use glucose
• WHY?
• Glucose is nearly always available
• Can be used without modification
• SO
• Glucose enzymes are always available
• (constitutive)

82
What if glucose AND lactose are available?

• Does the cell still
• transcribe lac genes?

83
with lactose ?lac genes ON
84
with lactose ?lac genes ON

• BUT
• lac operon has a weak promoter

85
with lactose ?lac genes ON

• BUT
• lac operon has a weak promoter
• GENES ARE ONLY WEAKLY ON

86
with lactose ?lac genes ON

• BUT
• lac operon has a weak promoter
• GENES ARE ONLY WEAKLY ON

• Less wasteful when the cell can use glucose
  instead

87

• So in fact

88
With lactose
Lac Z
89
? repressor free
Lac Z
90
Lac Z
? genes WEAKLY ON
Lac Z
weak
91
Without lactose
Lac Z
92
Repressor bound
Lac Z
x
93
genes OFF
Lac Z
x
94
genes OFF
Lac Z
x
? genes WEAKLY ON
Lac Z
95
BUT WHAT IF THERES NO GLUCOSE?
THE CELL MUST RELY ON LACTOSE

• How does the cell increase weak transcription of
  lac genes?

96
ACTIVATORpositive control
97
ACTIVATOR

• When glucose is low
• CAP binds CAP site

LacZ
LacY
LacA
CAP
O
98
ACTIVATOR

• When glucose is low
• CAP binds CAP site
• Actively recruits RNA polymerase

RNA pol
LacZ
LacY
LacA
O
CAP
99
ACTIVATOR

• When glucose is low
• CAP binds CAP site
• Actively recruits RNA polymerase

RNA pol
LacZ
LacY
LacA
O
CAP
100
ACTIVATOR

• When glucose is low
• CAP binds CAP site
• Actively recruits RNA polymerase

RNA pol
LacZ
LacY
LacA
O
CAP

• Great increase in transcription

101
POSITIVE CONTROLaccelerator down
102
Only want CRP to bind when glucose is unavailable

• How is CRP binding regulated by glucose?

103
cAMP
104
cAMP
inactive
active
105
CAP

• binds CAP site
• Actively recruits RNA polymerase

RNA pol
LacZ
LacY
LacA
O
CAP

• Great increase in transcription

106
ACTIVATOR

• cAMP Receptor Protein (CRP)
• Also known as
• Catabolic Activator Protein (CAP)

107
ACTIVATOR

• Gene encoding CAP is not near the lac genes

108
ACTIVATOR

• Gene encoding CAP is not near the lac genes
• DNA-binding protein activated by cAMP

109
ACTIVATOR

• Gene encoding CRP is not near the lac genes
• DNA-binding protein activated by cAMP
• Activates a number of different operons / genes
  that have upstream CAP site like sequences

110
WARNING!!

• The following slides may spoil your learning!
• They contain a summary of the regulation of the
  lac operon
• You will learn this material better if you make
  YOUR OWN summary, before you look at these slides!

111
SUMMARY
112
Bound to operator
113
TRANSCRIPTION
Bound to operator
114
TRANSCRIPTION
Bound to operator
OFF
115
TRANSCRIPTION
Bound to operator
OFF
116
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
117
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on
118
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
119
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
120
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
Released by allolact
121
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
Released by allolact
122
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
?cAMP
Released by allolact
123
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
?cAMP

• cAMP binds CRP
• CRP binds CAP site

Released by allolact
124
TRANSCRIPTION
Bound to operator
OFF
Released by allolactose
WEAKLY on (weak promoter)
?cAMP

• cAMP binds CRP
• CRP binds CAP site

Released by allolact
STRONGLY ON
125
Thus endeth the saga!
126
without lactose

• Lac repressor is bound to the operator
• RNA polymerase cannot bind to the promoter
• Transcription of lac genes is blocked

Lac Z
x
127
with lactose

• Allolactose binds the repressor
• RNA polymerase can bind the promoter
• Transcription of lac genes weakly ON