BIOL 2112 Chapter Notes - Chapter 18: Corepressor, Operon, Repressor

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Lecture 24 - Bacterial Gene Regulation

1) Bacteria respond to changes in their environments, such as nutrient

availability, by regulating their metabolic pathways. Describe two

mechanisms that bacteria use in order to adapt to environmental changes.

Include in your answer which mechanism is a short-term or long-term

adaptation. Feedback inhibition (anabolic biosynthetic pathways) allows a cell to adapt

to short term fluctuations in the supply of a substance it needs. They can do this by

adjusting to the activity of the enzymes already present which is a rapid response. This

response relies on chemical cues that increase or decrease their catalytic activity. Activity

of the first enzyme in the pathway is inhibited by the pathways end product. The end

product shuts down the synthesis of further production of the end product by inhibiting

the enzymes activity. Regulation of gene expression of genes encoding certain enzymes

allows a cell to adjust the production level of certain enzymes. If the environment provides

a molecule, the cell stops making the enzymes that catalyze the synthesis of that

molecule. The control of enzyme production occurs at the level of transcription in the

synthesis of mRNA from the genes that code for these enzymes. This is a long-term

response. 2) What is an operon and what is the advantage to this gene

structure? What is an operator sequence and where is it located within an

operon? An operon is the entire stretch of DNA required for enzyme production. Genes of

related function grouped into one single transcription unit are coordinately controlled by a

single operator. The operator is a o-off swith loated withi the prooter site

where RNA polymerase can bind to DNA and begin transcription) or between the

promoter and the enzyme-coding genes. The operator controls access of RNA polymerase

to the genes. The operon is constituted by the operator, the promoter, and the genes they

control. An operon is in the genome and there are many operons in a genome. 3) In

terms of an operon, what is a repressor and corepressor, and how do these

two factors function? An operon can be switched off by a protein called a repressor. A

repressor binds to the operator and blocks attachment of RNA polymerase to the promoter

preventing transcription of the genes. A repressor protein is specific for the operator of a

particular operon. A repressor for one operon has no effect on other operons in the

genome. Repressor proteins are encoded by a regulatory gene located some distance from

the operon and have each their own promoter (site where RNA polymerase can bind to

DNA and begin transcription). Regulatory genes are expressed continuously at a low rate

& a few repressor molecules are always present (in E. coli cells). Binding of repressors is

reversible and an operator alternates between two states – repressor bound & no

repressor bound. The relative duration of the repressor-bound state is higher when more

active repressor molecules are present. Most repressors (regulatory proteins) are allosteric

proteins, having two alternative shapes – active & inactive. The repressor is synthesized in

the inactive form which has little affinity for the operator. Only when a small molecule

cooperates with a repressor protein – binding to an allosteric site, therefore changing the

repressor protein to its active form – can the repressor attach to the operator turning the

operon off. In this type of system, the small binding molecule that cooperates with a

repressor protein to switch an operon off is called a corepressor. As the corepressor

accumulates, more corepressors associate with repressor molecules and then

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Document Summary

Lecture 24 - bacterial gene regulation: bacteria respond to changes in their environments, such as nutrient availability, by regulating their metabolic pathways. Describe two mechanisms that bacteria use in order to adapt to environmental changes. Include in your answer which mechanism is a short-term or long-term adaptation. Feedback inhibition (anabolic biosynthetic pathways) allows a cell to adapt to short term fluctuations in the supply of a substance it needs. They can do this by adjusting to the activity of the enzymes already present which is a rapid response. This response relies on chemical cues that increase or decrease their catalytic activity. Activity of the first enzyme in the pathway is inhibited by the pathways end product. The end product shuts down the synthesis of further production of the end product by inhibiting the enzymes activity. Regulation of gene expression of genes encoding certain enzymes allows a cell to adjust the production level of certain enzymes.

Related Questions

HELP ASAP;

Please help me understand my lab results, so be detailed to clearly understand . INclude answers to these questions what role does ampicilin (amp) and arabinose (ara) play in the experiment. Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid

GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes).

In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes.

The pGLO plasmid (Fig. 5, not a naturally occurring plasmid) contains the gene for GFP, and also contains the gene for the enzyme Î²-lactamase (bla), which provides resistance to the antibiotic ampicillin. The Î²-lactamase protein is produced and secreted by bacteria that contain the plasmid. Î²-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express Î²-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cellsâ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

for the first week section of lab

To each of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate.

Transfer this colony to one of the microfuge tubes. Gently vortex the tube until the colony is dispersed. Repeat for the second tube.

Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes.

Incubate both tubes at 42Â°C for 50 seconds.

Incubate both tubes on ice for 2 minutes.

Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes.

Flick each tube gently. Add 100ul of this transformation mix to the appropriate plates.

Results:

+DNA: LB/amp--> 1 colony, Glow: uncertain

+DNA: LB/amp/ara--> 9 colonies, Glow: yes

-DNA: LB/amp--> 7 colonies, GLow: no

-DNA: LB--> many colonies, Glow: no

HELP ASAP; Please help me understand my lab results, so be detailed to clearly understand

Then , Please include answers to the question below to include in your response

1. what role does ampicilin (amp) and arabinose (ara) play in the experiment.

2) Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes). In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes. The pGLO plasmid contains the gene for GFP, and also contains the gene for the enzyme Î²-lactamase (bla), which provides resistance to the antibiotic ampicillin. The Î²-lactamase protein is produced and secreted by bacteria that contain the plasmid. Î²-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express Î²-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cellsâ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells âcompetentâ (i.e. the cells are capable, or competent, to take-in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. 3) Cells are allowed to grow in a ârecoveryâ phase in the absence of ampicillin. During this time the cells begin to produce the Î²-lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin.

For the first week section of lab

To eac

h of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate. Transfer this colony to one of the microfuge tubes.

Gently vortex the tube until the colony is dispersed. Repeat for the second tube. Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes. Incubate both tubes at 42Â°C for 50 seconds.

Incubate both tubes on ice for 2 minutes. Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes. Flick each tube gently. Add 100ul of t

his transformation mix to the appropriate plates.

Results For plates:

+DNA: Has the E. Coli pGlo plasmid and - DNA plates do no have EColi pGLo plasmid .

Amp means ampiclin and Ara means arabinose on agar plates

+ DNA Plate LB/amp/ara--> white 9 colonies, Glow: yes

+DNA LB/amp--> 7 colonies, GLow: no

--DNA LB/AMP- 1 colony so no colony growth and no glovw

-DNA: LB--> many colonies, Glow: no

Please explain significance of result or thand the reason for the results in scientific terms based on the background information provided and your own knowledge to be detailed. Would these results be expected as wll.

1. Select which of the following are mechanisms used by pathogens to avoid host defenses and to successfully colonize the host.

A. Producing siderphores to obtain iron from the host for growth

B. producing adhesins for damaging host cells

C. Avoiding IgA proteases for avoiding secretory IgA

D. Avoiding secretory IgA by rapid turnover of pili

E. Producing toxic substances to suppress host responses

2. People considered obese have a different normal flora in the gastrointestinal tract compared to people who are considered lean.

A. true

B. false

3. Which of the following is NOT a mechanism used by pathogens for avoiding or surviving phagocytosis?

A. Killing macrophages and neutrophils

B. Colonizing the phagocyte

C. inhibiting opsonization

D. Inhibiting phagocte chemotaxis

E. these are all methods of avoiding or surviving phagocytosis

4.Which of the following immune responses is best suited to prevent a virus from infecting a cell?

A. complement cascade

B. inflammation

C. Virus-specific antibodies

D. Fever

E. phagocytic cells

5. Which of the following is NOT a method used by pathogens for penetrating skin or mucous membranes?

A. Injection of effector proteins that induce endocytosis

B. Exploiting antigen-sampling processes

C. these are all mechanisms for penetrating skin or mucous membranes

D. Exploiting an injury

E. Production of endotoxin

6. Antibodies, which are produced as part of the adaptive immune response, can sometimes be damaging rather than beneficial. People suffering from an autoimmune disease called antiphospholipid antibody syndrome (APS) make antibodies that bind to phospholipids. Some of the signs and symptoms affected individuals experience are blood clots, stroke, heart attack, and kidney damage. What is the likely cause of these symptoms?

A. the anti-phospholipid antibodies cuse immune complexes that lodge in the affected organs

B. The person fails to make protective antibodies

C. The anti-phospholipid antibodies weaken certain organs that contain fat

D. the person makes antibodies that bind inappropriately to bacterial capsules, preventing phagocytosis of these cells

E. Cell damage caused by binding of these antibodies initates inappropriate coagulation events which inferfere with blood flow in affected organs.

7. Only Gram-positive bacteria produce exotoxins

A. true

B. False

8. Which of the following members of the normal microbiota inhibit the growth of Candida albicans?

A. Lactobacilus species

B. Staphylococci species

C. E. coli

D. Neisseria species

E. Propionibacterium species

9. Would an antibody response against the B subunit of an A-B toxin protect against the effects of the toxin?

A. yes. if antibodies bind to the B protion, the toxin can no longer bind to target cells and will thus not affect those cells

B. No, even if antibodies bindto the B portion of the toxin, it is the A portion that actually causes damage to the cell

C. This question cannot be answer. The immune system does not mount a strong response against proteins, so this situation is unlikely to occur.

D. Yes, if antibodies bind to the B protein, that portion of the toxin does not become activated, and thus does not damage the host cell

E. No, even if antiibodies bind to the B portion of the toxin, the toxin is still taken into the host cell by phagocytosis, damaging that cell

10. Please select the correct definition of balanced pathogenicity.

A. A host-parasite relationship in which the parasite persists in the host, causing maximal harm. The pathogen becomes more virulent while the host becomes increasingly susceptible.

B. A mechanism that allows bacteria to transfer gene products directly into host cells, inducing changes such as altering the cell's cytoskeleton structure

C. A generated balance in the microbiome that may be caused by taking antimicrobial medication. it generally helps the host maintain optimal health

D. A situation in which an abnormally high number of T-helper cells are stimulated, causing a massive release of cytokines.

E. A host-parasite relationship in which the parasite persists in the host while causing minimal harm. The pathogen becomes less virulent while the host becomes less susceptible

11. Bacteria may survive phagocytosis by

A. preventing encounters with phagocytes AND preventing fusion of two pathogosomes

B. avoiding recognition and attachment of lymphocytes AND lysinf the phagosomes

C. preventing fusion of the lysosome with the phagosome AND mimicking host molecules

D. lysing the phagosome AND mimicking host molecules

E. preventing fusion of the lysosome with the phagosome AND lysing the phagosome

BIOL 2112 Chapter Notes - Chapter 18: Corepressor, Operon, Repressor

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