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Biology 22
Operons
REGULATION OF GENE EXPRESSION IN PROKARYOTES
Follow diagrams in your textbook and notes from the lecture to construct each of the
operon models described below.
A. CONSTITUTIVE SYSTEMS (e.g. general metabolic enzymes)
1. Construct a model system:
Use yellow pop beads to represent 4 structural genes.
Use red pop beads to represent the mRNA transcribed from these
structural genes.
Use blue pop beads to represent the protein enzymes translated
from the mRNA.
Use 11 red pop cylinders to represent the promoter.
Use the wooden block labeled "RNA polymerase" to represent that enzyme.
2. Set up the model and run it through all steps:
Have the polymerase attach to the promoter, then pass along the
structural genes to produce mRNA. Have the mRNA separate from
the DNA and use it to synthesize proteins.
3. Consider what would happen if each of the following were to occur:
a mutation removed half of the promoter region.
one base was substituted in the middle (-25) of the promoter region.
five bases were added to the middle of the promoter region.
one nucleotide was substituted at position –35.
the start codon was deleted from the first structural gene.
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B. The Lactose Operon: An Inducible Operon under both Negative and Positive
Control
The product of the regulatory gene I is an active repressor that binds to the
operator in the absence of lactose. When lactose binds to the repressor, the
repressor can no longer block the path of RNA polymerase from the promoter to
the structural genes. Positive control is exerted by the cAMP + CAP complex.
CAP is produced in an inactive form. When glucose levels are low, cAMP builds
up. CAP is activated by cAMP binding and the complex attaches to the promoter
to enhance transcription.
1. Construct a model of the operon:
Use yellow pop beads to represent the three structural genes: lacZ, lacY, lacA.
Use red beads to represent the mRNA and blue beads to represent the protein
gene products.
Use 9 blue pop cylinders to represent the operator.
Use 6 red pop cylinders to represent one portion of the promoter and one red pop
cylinder with a white glass rod to represent the promoter region called CBS (CAP
binding site) where the CAP + cAMP complex attaches.
Use the tubing labeled cAMP to attach to the tubing labeled CAP. In order to
enhance RNA polymerase binding, cAMP + CAP must bind to the CBS region of
the promoter. Remember that cAMP and glucose concentrations are
inversely proportional.
Use 8 yellow pop cylinders for the regulatory gene (I) and red pop beads for its
mRNA.
Use the wooden block labeled "active repressor protein" for the regulatory gene
product. Note how the lactose block can be used to alter its shape.
2. Show how the operon works under the following conditions:
a. High lactose and high glucose in the environment.
b. High lactose and low glucose in the environment.
c. Low lactose and high glucose in the environment.
d. Low lactose and low glucose in the environment.
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B. The Lactose Operon (continued)
3. Consider the effects of mutations in each of the regions of this operon:
the promoter for the structural genes is deleted.
the operator region is deleted.
a mutation in the lacI gene such that the gene product can no longer bind to
lactose.
the start codon for the lacZ gene is deleted.
a mutation in CAP such that it can bind to cAMP but the complex can no
longer bind to the promoter for the lac structural genes.
4. If a fourth structural gene were inserted after the third one by genetic
engineering techniques, what cellular conditions would lead to its
being expressed? Would the identify of the gene make any difference
(i.e. whether or not it were related to lactose in its activity)? Would it
make any difference, if it were a eukaryotic gene such as insulin,
whether the introns had been removed from the gene?
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C. The Arabinose Operon: Inducible Operon under both Positive and Negative
Control
For the arabinose operon, the same protein, the product of the araC gene, exerts
both negative and positive control. The araC product is a negative regulator
(active repressor) when arabinose is not bound to it. AraC binds to both araI and
araO creating a looped DNA structure that prevents RNA polymerase from
attaching to the promoter. AraC is a positive regulator (enhancing transcription)
when it binds to arabinose. The arabinose + araC product complex binds to the
araI region containing the promoter for the araB, araA and araD structural genes
to stimulate transcription.
This operon is also under the positive control of the cAMP + CAP complex.
Transcription of the structural genes is optimized when both the arabinose + araC
and cAMP + CAP complexes are attached to their binding sites on the common
promoter for araB, araA and araD. Transcription of the structural genes is rare in
the absence of these positive regulators.
1. Construct a model of the operon:
Use yellow pop beads to represent the three structural genes: araB, araA and
araD. Use red pop beads for mRNA, and blue pop beads for proteins.
Use 6 red pop cylinders and one red pop cylinder with a white glass rod to
represent the promoter region called CBS (CAP binding site) where the CAP +
cAMP complex attaches.
Use 6 blue pop cylinders to represent the operator and 6 additional blue pop
cylinders to represent the I site on the promoter where araC + arabinose bind.
Use 8 yellow pop cylinders to represent the regulatory gene, araC.
Use red pop beads for the mRNA of the regulatory gene.
Use the wooden block labeled "control protein" to represent the
gene product of the regulator gene. Note that the arabinose block
can change the shape of the control protein.
2. Show how the operon works under the following conditions:
a. High arabinose and high glucose in the environment.
b. High arabinose and low glucose in the environment.
c. Low arabinose and high glucose in the environment.
d. Low arabinose and low glucose in the environment.
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3. Consider the effects of mutations on each part of this system: (in all cases,
assume the environment is high in arabinose and low in glucose)
deletion of the I region.
deletion of the O region.
mutation in the araC gene such that the product cannot bind to arabinose.
mutation in the araC gene such that the product can bind to arabinose but
the araC + arabinose complex cannot bind to the I region.
mutation in the CAP gene such that the product cannot bind to cAMP.
mutation of the I region so that it can no longer bind the arabinose + ara C
complex.
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D. TRYPTOPHAN OPERON: A REPRESSIBLE OPERON WITH ATTENUATION
The tryptophan operon contains an "attenuator" region in the leader sequence
between the operator and the first structural gene. The attenuator is transcribed
by RNA polymerase, but if tryptophan is present, it interacts with the new
transcript fragment and causes the polymerase to fall off the DNA and
transcription to stop. If the polymerase gets beyond the attenuator region and
into the first structural gene, the transcript fragment gets folded in such a way that
tryptophan can no longer interact with it.
1. Construct a model system:
Use yellow pop beads to make five structural genes: trpE, trpD, trpC, trpB, trp A.
Use red and blue pop beads for mRNA and proteins.
Use 9 blue pop cylinders to represent the operator.
Use 6 red pop cylinders to represent one portion of the promoter. Attach 6 white
pop cylinders to represent the L region containing the attenuator.
Use 8 yellow pop cylinders to represent the regulatory gene.
Use red pop beads to represent the mRNA of the regulatory gene.
Use the wooden block labeled "repressor protein" to represent the product of the
regulatory gene.
Use the small wooden block labeled "co-repressor" to represent tryptophan. Note
how this block can be used to alter the shape of the "repressor protein" block. The
repressor protein is active only when it is bound to tryptophan.
Describe the conditions under which tryptophan can regulate the rate of
transcription of this operon.
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2. Use the model to illustrate how this operon carries out transcription
and translation under the following conditions:
High tryptophan in the environment.
Low tryptophan in the environment together with rapid protein
synthesis in the cell (uses tryptophan up).
Rapid synthesis of tryptophan with a low rate of protein synthesis in
the cell (does not use tryptophan at a rapid rate).
How many "start transcription" signals are there in this operon?
How many "stop transcription" signals?
How many signals are there to start and stop translation?
3. Consider events that could disrupt this operon such as:
What mutations in the operator could make this operon constitutive?
Could shut it off permanently?
What mutations in the promoter could alter the rate of transcription?
How serious would each one probably be?
How would mutations in the regulator gene affect the operon?
How would mutations in the structural genes affect the operon?
Which mutations would potentially have the greatest effect on the
ability of the bacterium to survive?
4. Tryptophan levels in the cell are also controlled by feedback inhibition, where
tryptophan inhibits the activity of the enzyme encoded by the trpE and trpD
genes. When tryptophan levels build up in the cell, which type of control would be
exerted first, feedback inhibition or repression of tryptophan operon transcription?
Be sure that you can explain your answer.
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