study guide/outline—bacterial gene regulation bacterial gene regulation what is an operon? how is...
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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?
Brooker Fig 16.3b
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
HH
H
H
OH
OHHO
HO
CH2OHO
HOH
Galactose
H
H
H
OH
OHH
CH2OHO
HOH
HH
H
H
OH
HO
CH2OHO
HOHH
H
H
O
OHH
CH2OHO
OHOH
HH
H
H
OH
HO
HO
CH2OHO
HOHH
H+
H+
H
H
OH
OHH
CH2
OO
HOH
Glucose
+
galactosidase
galactosidase
βgalactosidaseside reaction
Allolactose
Lactose
Cytoplasm
LactoseLactose permease
Functions of lactose permease and -galactosidase
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Brooker Fig 16.8
PromoterCAP site Operator
(b) No lactose or glucose (high cAMP)
cAMP
Transcription is very lowdue to the binding of therepressor.
Repressor
CAP
cAMP
CAP
PromoterCAP site Operator
Repressor(inactive)
High rate of transcription
Allolactose
Binding of RNA polymeraseto promoter is enhancedby CAP binding.
(a) Lactose, no glucose (high cAMP)
Positive control—Catabolite Activator Protein (CAP) turns on Lac Operon
High rate of transcription
But negative control Must be removed before positive control will result in transcription
Brooker, Fig 16.8
Allolactose
PromoterCAP site Operator
Repressor(inactive) (Inactive)
(Inactive)
(c) Lactose and glucose (low cAMP)
PromoterCAP site Operator
(d) Glucose, no lactose (low cAMP)
Transcription rate is lowdue to the lack of CAPbinding.
CAPTranscription is very low dueto the lack of CAP binding andthe binding of the repressor.
CAP
In absence of cAMP, transcription is very low (or hardly at all)
lac repressor bindsto the operator andinhibits transcription.
lacregulatorygene
lac operon
mRNA
lacI lacP lacO lacZ lacY lacA
lac repressor(active)
(a) No lactose in the environment
Figure 16.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Constitutive expression of
lacI
Promoter Operator
RNA pol cannot access the promoter when repressor bound to operator
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Figure 16.4
RNA polymerase
mRNA
lacI lacP lacO lacZ lacY lacA
Allolactose
Transcription
(b) Lactose present
galactosidase Lactosepermease
Galactosidetransacetylase
The binding of allolactose causes aconformational change that preventsthe lac repressor from binding to theoperator site.
Conformationalchange
PolycistronicmRNA
Lactose causes repressor to fall off Operator Site
Figure 16.5a
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Lac repressor
Lactose permease
galactosidase Lactose
Lac repressor
Transacetylase
4. Most proteins involvedwith lactose utilizationare degraded.
1. When lactose becomesavailable, a small amount of itis taken up and converted toallolactose by β-galactosidase.The allolactose binds to therepressor, causing it to fall offthe operator site.
2. lac operon proteinsare synthesized. Thispromotes the efficientmetabolism of lactose.
3. The lactose is depleted.Allolactose levels decrease.Allolactose is released fromthe repressor, allowing it tobind to the operator site.
Lac repressor
lac
operon
lac
operon
lac
operon
lac
operon
Induction of Lac Operon
Animation Lac Operon
http://vcell.ndsu.nodak.edu/animations/
http://vcell.ndsu.nodak.edu/animations/
Brooker Figure 16.7
4. Incubate the cells long enough to allow lac operon induction.
5. Burst the cells with a sonicator. Thisallows β-galactosidase to escape fromthe cells.
– LactoseF’
F
In mero-zygote strain, the lac I+ gene on the F´ factormakes enough repressor to bind to both operator sites (restoring WT phenotype on main chromosome).
Lactose is taken up, is converted toallolactose, and removes the repressor.
1 2 3 4
Lactose
3.
+ Lactose
4.Z+
I–
P O Y+
A+ Z+
PO Y+
I+
Experimental level Conceptual level
1. Grow mutant strain and merozygote strain separately.
2.Divide each strain into two tubes.
3. In one of the two tubes, add lactose.
Mutantstrain
Merozygotestrain Merozygote
– Lactose
Mutant
F′
Operon is constitutive-on in Mutant strain becauseno repressor is made.
1.
+ Lactose
2.
Z+
I–
P O Y+
A+
Z+
I–
P O Y+
A+ Z+P O
Y+
A+I+
Z+
I–
PO Y +
A+
Z +
I–P O Y +
A +
A+
Z+
I–
P O Y+
A+ Z+P O Y+
I+ A +
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6. Add β-o-nitrophenylgalactoside (β-ONPG). This is a colorlesscompound. β-galactosidase willcleave the compound to producegalactose and o-nitrophenol (O-NP).O-NP has a yellow color. The deeperthe yellow color, the moreβ-galactosidase was produced.
7. Incubate the sonicated cells toallow β-galactosidase time tocleave β-ONPG.
8. Measure the yellow color producedwith a spectrophotometer. (Seethe Appendix for a descriptionof spectrophotometry.)
o-nitrophenyl-galactoside
O-NP
-galactosidase
Broken cell
1.
1 2 3 4
NO2NO2
+
2.
NO2NO2
+
3.
NO2
4.
NO2
+
ONPG Galactose
NO2
Brooker Figure 16.7, cont
Table 16.1 16 – 34
Question
Will a loss-of-function mutation in Plac (promoter sequence) be cis-dominant or trans-dominant?
Lactose status(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 BoundNo
ExpressionNo
ExpressionNo
Expressionnone
Present WT + Inactivated Open WT B-GalWT
PermeaseWT
Transacet.none
Present Lac Ymiss
Present Lac ZNons
Present P Lac(-)
Absent Lac Oc
Present Lac Oc
Lac Z
Lactose status
(assume absence of Glucose)
GenotypePromoter
Seq Repressor Operator
Seq Lac Z LacY Lac A
Type of mutation (e.g. cis-
dominant)
Absent Lac I (-)
AbsentF’-Lac I (+)
Lac I (-)
AbsentF’-LacOc
Lac O+
PresentF’-LacOc
Lac O+
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