Download model 1 - Instructure

Gene Regulation in Bacteria, SP15
A POGIL exercise developed by Dr. A. Schivell
Nemhauser/Crowe
MODEL 1:
The model shows the beginning of a regulated gene in E. coli. It is shown under four different
conditions and the resulting rates of transcription are reported.
A + B are "transcription regulator proteins"
Condition:
Ignore until Q 5
sigma
A: Glucose + Lactose
Amount of
transcript
(compared to maximum)
5%
RNA polymerase
B: Glucose only
C: Glycerol only
0.5%
A
B
0.5%
100%
D: Lactose only
1. Looking at these four cases, answer the following questions:
a. Which transcription regulator (A or B) helps activate transcription? A
b. Which transcription regulator (A or B) represses (blocks) transcription? B
c. Which transcription regulator (A or B) has more influence than the other? B
2. Suggest one molecular explanation for why transcription rates are not very high in condition
A. (Why don't sigma/RNA polymerase bind very often?)
The promoter is weak and rarely binds to the polymerase without assistance.
3. For effective regulation of the gene, should the transcription regulators have specific DNA
sequences that they can bind, or should they be able to bind any sequence of DNA? Explain.
They need to bind specific sequences so that they are in the right spot to help or hinder
transcription initiation
4. Would transcription regulator A work as efficiently if it bound where B does? Vice versa?
No, especially as B blocks transcription – it would not work well if it came before the
promoter
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Gene Regulation in Bacteria, SP15
A POGIL exercise developed by Dr. A. Schivell
Nemhauser/Crowe
5. Each "condition" in Model 1 is a different food source for the bacteria. Bacteria consume
carbohydrates (or ‘sugars’) from their environment to make cellular energy. Lactose, galactose,
glycerol and glucose are all sugars that might be consumed by a bacteria.
Fill in the following sugars in their corresponding blanks in Model 1. The transcribed gene
codes for the β-galactosidase enzyme (this enzyme breaks lactose into glucose and galactose
sugars).
Condition A: Glucose and Lactose
Condition B: Glucose alone
Condition C: Glycerol alone
Condition D: Lactose alone
a. Why should a sugar source influence the production of β-galactosidase?
The sugar presence or absence is the cue which allows the cell to respond appropriately to
the environment. If the sugar was not used, the gene would not respond at the right times
and might be non-functional or wasteful.
b. Which transcription regulator is responding to the presence of glucose? A
Explain your answer in one grammatically correct sentence.
When glucose is present, then A is not bound to DNA and vice versa.
c. Which transcription regulator is responding to the presence of lactose? B
Explain your answer in one grammatically correct sentence.
When lactose is present, then B is not bound to DNA and vice versa
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Gene Regulation in Bacteria, SP15
A POGIL exercise developed by Dr. A. Schivell
Nemhauser/Crowe
MODEL 2:
Transcription regulators A and B both have two mutually distinct conformations based on the
binding of a small molecule:
DNA-binding
Conformation
(binds DNA)
Cannot bind
DNA
cAMP
A
lactose
B
6. a. In what state does A bind to DNA? when cAMP is bound
Note: bound = attached
when cAMP is not bound
b. What is the logical relationship between glucose levels and cAMP levels in a cell?
When glucose is high, cAMP is low and vice versa
7. a. In what state does B bind to DNA? when lactose is bound
when lactose is not bound
b. Explain how B's response to lactose is logical given how B regulates the production of the
enzyme β-galactosidase.
When lactose is present, it removes the transcription repressor, allowing the enzyme that
breaks it down to be synthesized
8. A and B proteins are encoded by their own genes elsewhere on the E. coli chromosome. For
effective regulation, should the A and B genes be constitutively expressed or be regulated?
Explain
Constitutively. Because you need them all the time as "sensors" for the various sugars
9. Imagine that the gene coding for regulator B was mutated so that the B protein's DNA
binding site was deleted completely. Estimate β-galactosidase enzyme production for each
scenario in Model 1 for this mutant.
A: 5%
B: 5%
C: 100%
D: 100%
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Gene Regulation in Bacteria, SP15
A POGIL exercise developed by Dr. A. Schivell
Nemhauser/Crowe
Post-Activity Information:
For this activity, I have removed the names of various proteins and gene sequences to make it
simpler. However, this model system is extensively studied and comes with many new names
that you'll have to learn for lab and problem sets/exams. Here are the "real" names:
- The β-galactosidase protein in bacteria is actually coded for by a gene called LacZ that is part
of a unique construct called an "operon". We'll discuss the structure of the "Lac operon" in lab.
However, the fact that this gene is part of an operon has no inherent effect on how it is
regulated. The regulation scheme in this activity could be for any gene, whether a part of an
operon or not.
-Transcription regulator A is the "Catabolite Activator Protein" (CAP). It binds to the "CAP
binding site" or CBS DNA sequence just upstream of the promoter of the β-galactosidase gene.
CAP is encoded by its own gene (the CAP gene).
- Transcription regulator B is the Lac Repressor protein. The gene that encodes the lac
repressor is called LacI. The lac repressor binds to the operator sequence (also known as
LacO) that is downstream of the promoter of the β-galactosidase gene. The operator sequence
spans the +1 site, effectively blocking transcription if the repressor is bound.
On Your Own:
1. LacOC is a version of the operator that cannot bind the repressor. LacIS is a version of the
repressor that cannot bind lactose, but can bind the operator.
You have an artificially diploid E. coli with two copies of the lac operon with the following
genotypes: (Assume that the CAP protein and CBS are normal)
lacI lacO lacZ / lacIS lacOC lacZ
a. In the drawing below of the two operons in this diploid cell, DRAW what is bound at both
promoters and operators under the condition of ONLY LACTOSE. Make sure to LABEL all
molecules you draw. (Include regulators as well as sigma/RNA polymerase.)
cAMP
cAMP
CAP
CBS
CAP
CBS
RNA
pol/
promoter
sigma
RNA pol/
promoter
sigma
operatorC
lacZ
lacY
lacA
operator
repressorS
lacZ
lacY
lacA
b. For each food source, circle "+" to indicate some or lots of β-galactosidase activity in the cell
or "-" for little/none.
lactose: +
glucose: glycerol: +
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Gene Regulation in Bacteria, SP15
A POGIL exercise developed by Dr. A. Schivell
Nemhauser/Crowe
2. The following diagram depicts the “riboflavin operon” found in E. coli. RibG, RibB, and
RibA code for enzymes required for the synthesis of riboflavin. A separate gene, RibI, codes
for a repressor protein that binds to the operator.
promoter
operator
RibG
RibB
RibA
a. When do you expect the RibI gene to be expressed? (Circle ONE)
- in the presence of riboflavin
- in the absence of riboflavin
- constitutively
b. Under what condition would you expect to
find the Rib repressor protein bound to the operator? When riboflavin levels are HIGH
c. The repressor protein must bind DNA… (Circle one)
- at the same time as it binds a molecule of riboflavin.
- only when not bound to a molecule of riboflavin.
- whether or not riboflavin is bound to it.
d. You discover a mutant E. coli cell that cannot make riboflavin. Suggest one part of the
operon or its regulation that could be mutated and describe how the mutation creates the
problem with riboflavin synthesis. (1-3 sentences.)
-RibG (or RibB, or RibA) gene could be mutated making it non-functional
-RibI (repressor) gene could be mutated so that the repressor protein always bound DNA
3. In E. coli, the genes in the in Trp operon code for enzymes needed to synthesize tryptophan.
Normally, the Trp repressor protein regulates the operon by binding to the operator (O) only
when tryptophan levels are high.
Imagine a different species in which the Trp repressor protein had only one conformation bound to the operator. Using the diagram below if needed, draw and/or describe a new
mechanism by which this species would still express the Trp operon genes only when needed.
Trp repressor
gene
P
P
O TrpA TrpB
In this new species, if the Trp repressor could not change structure, it would mean that it
only has one conformation (which must bind DNA at the operator to be effective). That
subsequently means that the repressor gene itself must be regulated by the presence of
tryptophan. By adding a new "Trp repressor repressor" protein to the system and adding a
different operator sequence to the Trp repressor gene, you could still get regulation. (See
answer to #14 of the exercise for why this is not as "elegant" a system).
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