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?
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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,
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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?
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.
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.
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)