Transcript Bacterial Gene Expression—Lac Operon

Study Guide/Outline—Bacterial Gene Regulation
Bacterial Gene Regulation
• What is an operon? How is it different from a eukaryotic gene?
• In the lac operon, what cellular or environmental conditions must exist in
order for the (WT) lac operon to express its genes? How do these
environmental conditions positively or negatively regulate the operon?
• What are the different parts, and their functions, of the operon?
• How do mutations in “upstream” parts of the operon (promoter, operator,
coding genes) affect the “downstream” areas of the operon? How do
missense and nonsense mutations have different results?
• The lacI gene is not part of the Lac Operon. How is the lac I gene involved
with the Lac operon?
• What kinds of mutations are cis-dominant? Trans-dominant? Constitutive
ON? Constitutive-OFF?
• How can a bacteria be a partial diploid? How does being diploid for the LacI
gene create complexities in the regulation of the Lac Operon?
Functions of lactose permease and b-galactosidase
Lactose
H+
Lactose permease
H+
Cytoplasm
CH2OH
CH2OH
O
O OH
HO
H
H
Lactose H
O
OH
H
OH
H
H
H
H
H
OH
H
β-galactosidase
side reaction
O
HO
b-galactosidase
H
CH2OH
O
H
OH
H
O
CH2
O OH
H
H
HO
H
OH
CH2OH
CH2OH
H
OH
H
O OH
O OH
H
H
H
+
b-galactosidase
OH
H
OH
H
H
H
H
HO
H
Allolactose
H
OH
HO
H
OH
Galactose
H
OH
Glucose
Brooker Fig 16.3b
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Positive control—Catabolite Activator
Protein (CAP) turns on Lac Operon
CAP site
Promoter
Operator
Allolactose
cAMP
High rate of transcription
High rate of transcription
CAP
Repressor
(inactive)
Binding of RNA polymerase
to promoter is enhanced
by CAP binding.
(a) Lactose, no glucose (high cAMP)
CAP site
Promoter
Operator
Repressor
cAMP
CAP
Brooker Fig 16.8
Transcription is very low
due to the binding of the
repressor.
(b) No lactose or glucose (high cAMP)
But negative control
Must be removed
before positive
control will result in
transcription
In absence of cAMP, transcription is very low
(or hardly at all)
CAP site
Promoter Operator
Allolactose
CAP
Repressor
(inactive)
Transcription rate is low
due to the lack of CAP
binding.
(Inactive)
(c) Lactose and glucose (low cAMP)
CAP site Promoter
Operator
CAP
(Inactive)
Transcription is very low due
to the lack of CAP binding and
the binding of the repressor.
(d) Glucose, no lactose (low cAMP)
Brooker, Fig 16.8
RNA pol cannot access the promoter when repressor bound to
operator
lac
regulatory
gene
lac operon
Promoter
lacI
Constitutive
expression of
lacI
lacP
Operator
lacO
lacZ
lacY
lacA
mRNA
lac repressor binds
to the operator and
inhibits transcription.
lac repressor
(active)
(a) No lactose in the environment
Figure 16.4
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
(b) Lactose present
Lactose
causes repressor to fall
off Operator Site
RNA polymerase
lacI
lacP
lacO
lacZ
lacY
lacA
Transcription
mRNA
Polycistronic
mRNA
b-galactosidase Lactose
permease
Galactoside
transacetylase
Allolactose
Conformational
change
The binding of allolactose causes a
conformational change that prevents
the lac repressor from binding to the
operator site.
Figure 16.4
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Induction of Lac Operon
Transacetylase
Lac repressor
b-galactosidase
1. When lactose becomes
available, a small amount of it
is taken up and converted to
allolactose by β-galactosidase.
The allolactose binds to the
repressor, causing it to fall off
the operator site.
Lac repressor
Lactose
Lactose permease
2. lac operon proteins
are synthesized. This
promotes the efficient
metabolism of lactose.
4. Most proteins involved
with lactose utilization
are degraded.
Lac repressor
3. The lactose is depleted.
Allolactose levels decrease.
Allolactose is released from
the repressor, allowing it to
bind to the operator site.
Figure 16.5a
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Animation Lac Operon
http://vcell.ndsu.nodak.edu/animations/
http://vcell.ndsu.nodak.edu/animations/
Experimental level
Conceptual level
Mutant
+
P O Z Y+
I–
1. Grow mutant strain and merozygote
strain separately.
Mutant
strain
Merozygote
strain
I–
A+
+ +
PO Z Y +
A
1.
P
I–
+
I+
P O ZY+
+
F′ A
O Z+ Y +
A+
2.Divide each strain into two tubes.
Merozygote
–
Lactose
Operon is constitutive-on in Mutant strain because
no repressor is made.
2.
P O Z +Y +
A+
I–
+
Lactose
–
Lactose
Lactose
3. In one of the two tubes, add lactose.
3.
I–
1
2
3
+ +
PO Z Y +
A
4
P O Z Y+
A+
F’
In mero-zygote strain, the lac I+ gene on the F´ factor
makes enough repressor to bind to both operator sites
(restoring WT phenotype on main chromosome).
4. Incubate the cells long enough to allow
lac operon induction.
5. Burst the cells with a sonicator. This
allows β-galactosidase to escape from
the cells.
+
I+
4.
PO
I–
Z+Y+
A+
P
I+
O Z+ Y+
+
F A
+
Lactose
Lactose is taken up, is converted to
allolactose, and removes the repressor.
Brooker Figure 16.7
6. Add β-o-nitrophenylgalactoside
(β-ONPG). This is a colorless
compound. β-galactosidase will
cleave the compound to produce
galactose and o-nitrophenol (O-NP).
O-NP has a yellow color. The deeper
the yellow color, the more
β-galactosidase was produced.
b-ONPG GalactoseO-NP
b-o-nitrophenyl- 1.
galactoside
+
NO2
NO2
Broken cell
b-galactosidase
+
2.
NO2
3.
7. Incubate the sonicated cells to
allow β-galactosidase time to
cleave β-ONPG.
NO2
1
2
3
4
4.
+
NO2
8. Measure the yellow color produced
with a spectrophotometer. (See
the Appendix for a description
of spectrophotometry.)
NO2
NO2
Brooker Figure 16.7, cont
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Table 16.1
16 – 34
Question
Will a loss-of-function mutation in Plac
(promoter sequence) be cis-dominant or
trans-dominant?
Lactose
status
Genotype
Promoter
Seq
Repressor
Operator
Seq
(assume
absence of
Glucose)
Lac Z
Lac Y
Lac A
Type of
mutation
(e.g. cisdominant,
consititutive
ON)
Lac Z
Absent
WT
+
Active
Bound
No
Expression
No
Expression
No
Expression
none
Present
WT
+
Inactivated
Open
WT B-Gal
WT
Permease
WT
Transacet.
none
Present
Lac Ymiss
Present
Lac ZNons
Present
P Lac(-)
Absent
Lac Oc
Present
Lac Oc
Lactose
status
(assume
absence of
Glucose)
Genotype
Absent
Lac I (-)
Absent
F’-Lac I (+)
Lac I (-)
Absent
F’-LacOc
Lac O+
Present
F’-LacOc
Lac O+
Promoter
Seq
Repressor
Operator
Seq
Lac Z
LacY
Lac A
Type of
mutation
(e.g. cisdominant)
Go over lecture outline at end of
lecture