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Gene Regulation (Prokaryotes) – LS1a: Final Exam Review
Cell Signaling->Gene Regulation:
Why regulate genes?
Prokaryotic v. Eukaryotic Gene Regulation:
- Multi-cellular organisms must regulate cell differentiation as well as respond to
changing environmental conditions.
E. coli. (7 sigma factors)
σ70 – responsible for 90% of genes
σH – ‘Heat Shock’: activated by high temperature environment
Repressors and Activators:
Repressor – binds to “operator” between -35 and -10, prevents RNA Pol from binding
and transcribing operon.
Activators - binds upstream of -35, interacts with POL and encourages transcription
from less ‘idealized’ promoters.
Jacob and Monod: The LAC Operon
Reporter: ONPG reports the activity of B-gal (and thus indirectly reports the
expression of lacZ)
LAC Operon: E. Coli.response to lactose-only environment
Diploid Analysis:
Determine Trans v. Cis…
Le Chatelier’s Principle: “a system at equilibrium subjected to ‘stress’ will
adjust to relieve the ‘stress’ and restore equilibrium.”
Q1. Bacterial cells can take up the amino acid tryptophan (Trp)
from their surroundings, or if there is an insufficient external
supply, they can synthesize tryptophan from other small
molecules. The Trp repressor is a bacterial gene regulatory
protein that shuts off the transcription of genes that code for the
enzymes required for the synthesis of tryptophan. Trp repressor
binds to a site in the promoter of these genes only when
molecules of tryptophan are bound to it (see figure below).
a) Why is this a useful property of the Trp repressor?
Q1 contd.
b) What would happen to the regulation of the tryptophan
biosynthesis enzymes in cells that express a mutant form of Trp
repressor that (1) cannot bind to DNA or (2) binds to DNA even if
no tryptophan is bound to it?
Q1 contd.
c) What would happen in scenarios (1) and (2) if the cells, in
addition, produced normal Trp repressor protein from a second,
normal gene?
Q2. In the absence of glucose, E. coli can proliferate using the
pentose sugar arabinose. The ability of E. coli to utilize the
sugar arabinose is regulated via the arabinose operon, depicted
in the figure below.
The araA, araB, and araD genes encode enzymes for the
metabolism of arabinose. The araC gene encodes a gene
regulatory protein that binds adjacent to the promoter of the
arabinose operon. To understand the regulatory properties of
the AraC protein, you engineer a mutant bacterium in which the
araC gene has been deleted and look at the effect of the
presence or absence of the AraC protein on the AraA enzyme.
a) If the AraC protein works as a gene repressor, would you
expect araA RNA levels to be high or low in the presence of
arabinose in the araC– mutant cells? What about in the
araC– mutant cells in the absence of arabinose? Explain your
answer.
Q2 contd.
b) Your findings from the experiment are summarized in the
following table:
Do the results indicate that the AraC protein regulates
arabinose metabolism by acting as a gene repressor or a gene
activator? Explain your answer.
Q3. Progesterone is a steroid hormone that prepares the
body for pregnancy. Progesterone exerts its function by
binding to the progesterone receptor in the cell. This binding
event in turn leads to the transcriptional activation of
various genes.
Progesterone production is stimulated by Luteinizing
Hormone (LH), whose production is stimulated by
gonadotropin releasing hormone (GnRH). This pathway can
be illustrated as shown below:
a) High levels of progesterone suppress the release of GnRH,
and in turn lead to a reduction in progesterone levels. What
term is used to describe this type of feedback control?
Q3 contd.
b) If this were an example of the opposite type of feedback
control, what effect would high levels of progesterone have
upon it’s own production?
Q3 contd.
c) The structures of progesterone and cholesterol are shown
below. Do you think progesterone requires a special
transporter to pass through the plasma membrane?
Explain.
Q3 contd.
Prior to progesterone binding, Progesterone Receptor (PR) is
predominantly found in the cytoplasm. However after
progesterone binds, the hormone/receptor complex moves
into the nucleus, binds to DNA, recruits transcription factors,
and activates transcription.
d) If Progesterone Receptor is unable to move into the
nucleus, could transcriptional
activation occur? Explain?
Q3 contd.
e) The lacZ gene can be used as a reporter to study
transcriptional activation, because its protein product (Βgalactosidase) can be easily measured by a simple enzyme
assay. The wildtype and a mutant PR were studied using the
DNA construct shown below. Progesterone Response
Element (PRE) refers to the DNA sequence that is bound by
the activated PR.
The following results were obtained, where the asterisk
indicates when progesterone was (or was not) added to the
cells. Please explain what has happened to the mutant
PR and how the data supports your interpretation.
Q1. Bacterial cells can take up the amino acid tryptophan (Trp)
from their surroundings, or if there is an insufficient external
supply, they can synthesize tryptophan from other small
molecules. The Trp repressor is a bacterial gene regulatory
protein that shuts off the transcription of genes that code for the
enzymes required for the synthesis of tryptophan. Trp repressor
binds to a site in the promoter of these genes only when
molecules of tryptophan are bound to it (see figure below).
a) Why is this a useful property of the Trp repressor?
In the presence of environmental TRP, bacteria should not waste resources
on its synthesis, but should respond to an absence of TRP by expressing the
biosynthesis machinery required for its production.
Q1 contd.
b) What would happen to the regulation of the tryptophan
biosynthesis enzymes in cells that express a mutant form of Trp
repressor that (1) cannot bind to DNA or (2) binds to DNA even if
no tryptophan is bound to it?
1. TRP biosynthesis machinery always active; no response to conditions.
2. TRP biosynthesis machinery always repressed; no response to
conditions.
Q1 contd.
c) What would happen in scenarios (1) and (2) if the cells, in
addition, produced normal Trp repressor protein from a second,
normal gene?
1. The WT TRP repressor works in trans to regulate both TRP operons
normally.
2. The mutant TRP repressor works in trans to constantly repress both
TRP operons, regardless of environmental conditions.
Q2. In the absence of glucose, E. coli can proliferate using the
pentose sugar arabinose. The ability of E. coli to utilize the
sugar arabinose is regulated via the arabinose operon, depicted
in the figure below.
The araA, araB, and araD genes encode enzymes for the
metabolism of arabinose. The araC gene encodes a gene
regulatory protein that binds adjacent to the promoter of the
arabinose operon. To understand the regulatory properties of
the AraC protein, you engineer a mutant bacterium in which the
araC gene has been deleted and look at the effect of the
presence or absence of the AraC protein on the AraA enzyme.
a) If the AraC protein works as a gene repressor, would you
expect araA RNA levels to be high or low in the presence of
arabinose in the araC– mutant cells? What about in the
araC– mutant cells in the absence of arabinose? Explain your
answer.
If the AraC protein acted as a gene repressor for the arabinose operon, araA
RNA
levels should be high in the presence or absence of arabinose when there is no
AraC protein around. In fact, the araA RNA levels should be high all the time,
regardless of the presence or absence of arabinose, since the AraA gene should
be transcribed under all conditions in the absence of AraC.
Q2 contd.
b) Your findings from the experiment are summarized in the
following table:
Do the results indicate that the AraC protein regulates
arabinose metabolism by acting as a gene repressor or a gene
activator? Explain your answer.
The results are consistent with AraC acting as a gene activator for the arabinose
operon. A gene activator must bind to the promoter regions of the arabinose
genes in order to stimulate their transcription. Thus, if the gene for the
regulatory
protein is deleted, the arabinose genes cannot be turned on.
Q3. Progesterone is a steroid hormone that prepares the
body for pregnancy. Progesterone exerts its function by
binding to the progesterone receptor in the cell. This binding
event in turn leads to the transcriptional activation of
various genes.
Progesterone production is stimulated by Luteinizing
Hormone (LH), whose production is stimulated by
gonadotropin releasing hormone (GnRH). This pathway can
be illustrated as shown below:
a) High levels of progesterone suppress the release of GnRH,
and in turn lead to a reduction in progesterone levels. What
term is used to describe this type of feedback control?
negative feedback – high levels of progesterone should inhibit additional
synthesis, production, or release of additional progesterone
Q3 contd.
b) If this were an example of the opposite type of feedback
control, what effect would high levels of progesterone have
upon it’s own production?
positive feedback – progesterone is stimulating the production of additional
progesterone
Q3 contd.
c) The structures of progesterone and cholesterol are shown
below. Do you think progesterone requires a special
transporter to pass through the plasma membrane?
Explain.
progesterone should be able to move through the membrane without a
transporter as it is mainly a hydrophobic molecule similar to the membrane
component, cholesterol.
Q3 contd.
Prior to progesterone binding, Progesterone Receptor (PR) is
predominantly found in the cytoplasm. However after
progesterone binds, the hormone/receptor complex moves
into the nucleus, binds to DNA, recruits transcription factors,
and activates transcription.
d) If Progesterone Receptor is unable to move into the
nucleus, could transcriptional
activation occur? Explain?
no. If the receptor cannot enter the nucleus, it would be unable to bind to
the genomic DNA and activate transcription.
Q3 contd.
e) The lacZ gene can be used as a reporter to study
transcriptional activation, because its protein product (Βgalactosidase) can be easily measured by a simple enzyme
assay. The wildtype and a mutant PR were studied using the
DNA construct shown below. Progesterone Response
Element (PRE) refers to the DNA sequence that is bound by
the activated PR.
The following results were obtained, where the asterisk
indicates when progesterone was (or was not) added to the
cells. Please explain what has happened to the mutant
PR and how the data supports your interpretation.
The mutant PR is constitutively active (always on) independent of
progesterone binding. One of the ways transcription is controlled is by
keeping transcriptional activators away from the DNA. However if a
transcriptional activator is now always by the gene’s they activate, such as
if
the progesterone receptor is always in the nucleus, it may be able to
activate
transcription all the time. [Note: other answers may be possible and would
be
given credit if properly explained.]