1. Study Guide/Outline—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?

2. Functions of lactose permease and b-galactosidaseH+
Lactose permease H+
Cytoplasm
CH2OH
CH2OH HO O H O OH
β-galactosidase
side reaction
Lactose H O H OH H OH H H H H H OH H O
CH2OH O
CH2 HO O H O OH
b-galactosidase H H
Allolactose OH H OH H H H HO H
CH2OH
CH2OH H OH H OH HO O OH H O OH H + H
b-galactosidase OH H OH H H H HO H H OH H OH
Galactose
Glucose
Brooker Fig 16.3b
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3. 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
Binding of RNA polymerase
to promoter is enhanced
by CAP binding.
Repressor
(inactive)
(a) Lactose, no glucose (high cAMP)
CAP site
Promoter
Operator
Repressor
cAMP
CAP
Transcription is very low
due to the binding of the
repressor.
But negative control
Must be removed before positive control will result in transcription
(b) No lactose or glucose (high cAMP)
Brooker Fig 16.8
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. In absence of cAMP, transcription is very low (or hardly at all)
CAP site
Promoter
Operator
Allolactose
Transcription rate is low
due to the lack of CAP
binding.
CAP
Repressor
(inactive)
(Inactive)
(c) Lactose and glucose (low cAMP)
CAP site
Promoter
Operator
CAP
Transcription is very low due
to the lack of CAP binding and
the binding of the repressor.
(Inactive)
(d) Glucose, no lactose (low cAMP)
Brooker, Fig 16.8
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

5. RNA pol cannot access the promoter when repressor bound to operator
lac operon
lac
regulatory
gene
Promoter
Operator
lacI
lacO
lacZ
lacY
lacP
lacA
Constitutive expression of lacI
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

6. Lactose causes repressor to fall off Operator Site
(b) Lactose present
RNA polymerase
lacI
lacZ
lacY
lacP
lacO
lacA
Transcription
Polycistronic
mRNA
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

7. Induction of Lac Operon
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.
Transacetylase
Lac repressor
b-galactosidase
Lactose
Lac repressor
lac operon
lac operon
Lactose permease
4. Most proteins involved
with lactose utilization
are degraded.
2. lac operon proteins
are synthesized. This
promotes the efficient
metabolism of lactose.
lac operon
lac operon
3. The lactose is depleted.
Allolactose levels decrease.
Allolactose is released from
the repressor, allowing it to
bind to the operator site.
Lac repressor
Figure 16.5a
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8. Animation Lac Operon

9. – + – + Brooker Figure 16.7 Experimental level Conceptual level
Mutant P O Z+ Y+ A+ I–
1. Grow mutant strain and merozygote
strain separately.
Mutant
strain
Merozygote
strain P O Z+ Y+ I– A+ P O Z+ I+ Y+ F′ A+
Merozygote 1. O Z+ P
Y + A+ – I–
Lactose
2.Divide each strain into two tubes.
Operon is constitutive-on in Mutant strain because
no repressor is made. 2. P O
Z +
Y + I –
A + +
Lactose
Lactose
3. In one of the two tubes, add lactose. 3. P O Z+ Y+ O Z+ – I– A+ P Y+ I+
A +
Lactose F’ 1 2 3 4
4. Incubate the cells long enough to allow
lac operon induction.
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 .
5. Burst the cells with a sonicator. This
allows β-galactosidase to escape from
the cells. P O Z+ Y+ A+ P O Z+ + Y+ I– I+ F A+
Lactose
Lactose is taken up, is converted to
allolactose, and removes the repressor.
Brooker Figure 16.7

10. Brooker Figure 16.7, cont 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
Galactose
O-NP
b-o-nitrophenyl-
galactoside 1 . +
NO2
NO2
Broken cell
b-galactosidase 2 . +
NO2
NO2 3 .
7. Incubate the sonicated cells to
allow β-galactosidase time to
cleave β-ONPG.
NO2 1 2 3 4 4 . +
NO2
NO2
8. Measure the yellow color produced
with a spectrophotometer. (See
the Appendix for a description
of spectrophotometry.)
Brooker Figure 16.7, cont
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11. Table 16.1
16 – 34

12. Question
Will a loss-of-function mutation in Plac (promoter sequence) be cis-dominant or trans-dominant?

13. + Lactose status Repressor Operator Seq Lac Y Type of mutation Absent
(assume absence of Glucose)
Genotype
Promoter Seq
Repressor
Operator Seq
Lac Z
Lac Y
Lac A
Type of mutation
(e.g. cis-dominant, consititutive ON)
Absent WT +
Active
Bound
No Expression
none
Present
Inactivated
Open
WT B-Gal
WT Permease
WT Transacet.
Lac Ymiss
Lac ZNons
P Lac(-)
Lac Oc

14. (assume absence of Glucose)
Lactose status
(assume absence of Glucose)
Genotype
Promoter
Seq
Repressor
Operator Seq
Lac Z
LacY
Lac A
Type of mutation
(e.g. cis-dominant)
Absent
Lac I (-)
F’-Lac I (+)
F’-LacOc
Lac O+
Present

15. Go over lecture outline at end of lecture