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 (Β-galactosidase) 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 (Β-galactosidase) 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.]