Gene Regulation in Prokaryotes''

1
S86
Gene Regulation in Prokaryotes.. And the
wonders of the Lac operon
RNA polymerase is a key protein, and regulation
of gene expression occurs frequently at
INITIATION.
2

• Lac operon
• Phenomenon
• On glucose- Bacteria grow fine
• On lactose- bacteria dont grow, then grow
  because they induce an enzyme that breaks lactose
  down into glucose
• b-galactosidase
• 3. ON lactose AND glucose- No b-galactosidase !
  

S87
Fig 16.3
3
How the Lac operon works. The Genes and
Regulatory elements
S88
Fig 16.10
resting state
repressed state
induced state
Lactose broken down to glucose!
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S89
A closer look.
Structural genes lacZ, lacY, lacA Alleles
lacZ lacY O P lacI Special guest
inducer lactose or IPTG
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S90
Molecular interpretations of special alleles
Fig 16.7
lacI
Oc
6
S91
DNA sequences determine if regulatory proteins
bind
lacI repressor
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S92
Use of F and bacterial genetics to study the lac
operon
Dont get acting in cis and trans confused
its actually a very simple concept
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S93
Table 1 Figuring Out the Lac Operon using Genetics
Phenotypes
b galactosidase (Z) permease (Y) Conclusion
Strain Genotype
No inducer inducer No inducer inducer
- -
wildtype
1a O Z Y
- - -
-
1b OZ-Y-
2a O Z Y/F OZY
- -
- - Z
Y dominant
2b O Z- Y-/F OZY

• - -
  Zdom.

2c O Z Y/F OZ-Y

• Oc
  .
  is constitutive

3. Oc Z Y

• - Oc
  . .
  acts in cis . and
  NOT in trans

4. OZ -Y/FOcZY-
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S94
Figuring out the Lac Operon using Genetics
Investigating I, the Repressor
Phenotypes
b galactosidase (Z) permease (Y) Conclusion
Strain Genotype
No inducer inducer No inducer inducer
- - wildtype
1 I Z Y
I- is
constitutive
2 I- Z Y
3 I Z- Y/F I-ZY

• - -

I dominant to I-

• - - I acts in
  tran .
  in cis.

4 I- Z- Y/F IZY-
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S95
Figuring out the Lac Operon using Genetics
Investigating Is, an allele of the Repressor
Phenotypes
b galactosidase (Z) permease (Y) Conclusion
Strain Genotype
No inducer inducer No inducer inducer
- - wildtype
1 I Z Y
- - - - IS
always represses
2 IS Z Y

• - - - -

IS dominant to I
3 IS Z Y/F I
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S97
An extra Test of IS and Oc.
Phenotypes
b galactosidase (Z) permease (Y) Conclusion
Strain Genotype
No inducer inducer No inducer inducer
1 ISOC Z Y-/F I-OZ-Y
2 ISO Z Y-/F I-OCZ-Y

• Fill in the phenotypes
• Does LacZ expression in Strain 1 tell you that I
  the repressor acts in trans? Why or Why not?
• Does LacY expression in Strain 1 tell you that I
  the repressor acts in trans? Why or Why not?
• What does LacY expression in Strain 2 tell you?

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S98

• Dual Regulation of the Lac operon

• Phenomenon
• On glucose- Bacteria grow fine
• On lactose- bacteria dont grow, then grow
  because they induce an enzyme that breaks lactose
  down into glucose
• b-galactosidase
• 3. On lactose AND glucose- No b-galactosidase !

Fig 16.11
Lets use bars and arrows to make this more
clear(board)
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S99

• 2 ways to understand the Lac operon
• See fig 16.10, S88
• Bars and Arrows

Adenyl Cyclase (ADC1)
cAMP
glucose
CRP
lacZ
Lactose (IPTG) (Inducer)
I
In this case, BOTH pathways must be satisfied
to permit lacZ expression
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• An Explanation of Bars and Arrows
• Key
• Is mutation recessive or dominant?
• Draw pathway with known genes, bars and arrows
• Use the following logic
• Example 1 for LacI
• Phenotype (lacZ)
• Genotype no inducer inducer
• 1. I -
• 2. I-1 (mutant)
• 3. I-1/I - I-1 recessive
• 4. I-2 - -
• 5. I-2/I - - I-2
  dominant
• Pathway inducer I Z

S100
2?
1?
Analysis- Bar or Arrow and I function? I-1 is
recessive- I-1 lost Is normal function. I-1
therefore does not do something that I normally
does Since I-1 yields Z, then I must normally
inhibit Z. BAR is right! (Also, imagine if I
were activator, and I-1 does not do that..then
I-1 would not make Z - which is not the phenotype
of I-1! ) I-2 is dominant I-2 does the same as
I, yet I-2 does it when I doesnt I-2-
activator of Z when I is not activating?? NO,
in I-2 no Z made I-2 is inhibitor of Z when I
is not an inhibitor? YES, in I-2 no Z. Bar is
right again!
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Gene F-1 problem
S101
Example 2 glu ADC1 CRP
lacZ
Genotype Phenotype (lactose present)
no glucose glucose 1.Gene F
(wildtype) - 2. Gene F-1
3. Gene F-1/Gene F 4. ADC1- -
- 5. ADC1-, Gene F-1 - -
Line 5 says that ADC1 is epistatic/downstream of
Gene F because double mutant has phenotype of
ADC1- single mutant
Bar or Arrow?
glu Gene F ADC1
CRP lacZ
Analysis Gene F-1 is dominant, so gene F-1
does something that Gene F normally does, yet
Gene F-1 does it when Gene F does not. Since in
Gene F-1 lacZ is made, (and all are arrows
downstream of ADC1), Gene F must also be an
activator. To maintain overall negative from
glucose, then bar is needed between glu and Gene
F. Note With Bars and arrows, start with most
downstream gene first, then work your way to
front!!
16
S102
Protein Binding Sites in the Lac Control Region
Fig 16.17, modified
-10 region
-35 region
TA
AT
OC mutations