1
Q
You have discovered base changes in the promoter region of the operon in a bacterial chromosome. Would you expect these changes to act in trans on another copy of the operon? Explain your reasoning.
A
- Mutations in promoters are not trans factors; the mutation can results in constitutive expression (always present) but should not act on other promoters
2
Q
What are cis- elements? What are trans- factors? Give an example from the Trp operon (or form the Ara-operon).
A
- cis element region of DNA/RNA that regulates expression of genes located on the same strand (e.g., trpO)- trans factor a molecule which regulates expression of genes distant from the gene that originally created it (e.g., repressor dimer)
3
Q
Draw the diagram of the lac operon that illustrates negative control
A
http://walkthemoonvevo.tumblr.com/image/163641843442
4
Q
You have isolated a protein that binds to DNA in the region upstream of the promoter sequence of the gene of interest. If this is a positive regulator (activator) which would be true:A) Loss of function mutation in the gene encoding this DNA binding protein would cause constitutive expressionB) Loss of function mutation in the gene encoding this DNA binding protein would result in lower or no expression.Explain your reasoning.
A
B –> since the molecule activates expression, loss of function mutation would result in lower expression
5
Q
Discuss why are lac Oc mutants cis-acting.
A
Oc is dominant over O+ so it results in bacteria always producing structural genes (constitutive) so bacteria always cleave lactose
* Cis acting since it is a mutation in the promoter O that results in the repressor being unable to bind
6
Q
Discuss why are lac I- mutants trans-acting.
A
- I- is a constitutive mutations that results in structural genes always being expressed
- Trans acting since mutated repressor that cannot bind to the promoter, therefore con
7
Q
Discuss positive and negative regulation of L-ara operon.
A
- The initiator region contains ara I, which codes for enzymes that act on arabinose
- ara C encodes the activator protein AraC
- When there is no arabinose, the AraC binds to ara O2 and ara I1 and bends DNA, hiding the promoter from RNA polymerase so enzymes are not synthesized
- Where arabinose is present, it binds to AraC, changing the conformation of the protein, so AraC binds to ara I1 and ara I2 making the promoter accessible to RNAP
8
Q
Regarding the regulation of Trp operon, what do we call the amino acid tryptophan? Why?
A
- Tryptophan is co-repressor
- It is necessary to activate the aporepressor protein that then binds to the promoter to negatively regulate synthesis of the structural genes
9
Q
What is meant by polycistronic mRNA? Give an example.
A
• mRNA that contains genes for more than one protein product (e.g., the lac operon)
10
Q
What is catabolite repression? What is the role of Catabolite Activator Protein? Explain its action. (remember the # of operons it activates!)
A
- Catabolite repression – the negative control of the expression of enzymes involved in catabolism of energy sources other than glucose; the “glucose effect”
- The Catabolite Activator Protein (CAP) or cyclic AMP (cAMP) will induce or active the pathway for synthesis of lactose by complexing with RNAP when cell is starving from lack of glucose
- cAMP binds to CAP, which binds to CAP binding site upstream from RNAP binding site, stimulating transcription
- called pleiotropic or general activator
11
Q
Define: repressor, co-repressor, aporepressor and inducer.
A
- Repressor – trans factors that bind to decrease the rate of transcription
- Co-repressor – activate aporepressors that are then capable of binding to decrease the rate of transcription
- Aporepressor – a repressor that cannot combine with an operator gene until it has combined with a corepressor
- Inducer – trans factors that inactive repressors thereby inducing transcription
12
Q
Define effector and inducer. Give example
A
- Effector – small molecules that change the conformation of repressors by binding; either inducers or corepressors
- Inducer – trans factors that inactive repressors thereby inducing transcription
13
Q
What are activators? What are enhancers?
A
- Enhancers – cis elements that, upon binding of an activator, recruits transcription factors that enhance the binding of RNA polymerase, thereby increasing the rate of transcription
- Activators – trans factors that act on enhancers
14
Q
What is the role of auxiliary operators?
A
- Multiple operators (auxiliary operators) can increase or decrease the level of transcription dramatically (e.g., 20-fold to 500-fold with 2 other operators in lac operon)
- Can increase the local concentration of the repressor or cause DNA looping as repressor binds to many operators which prevents binding of RNA polymerase
15
Q
Discuss the type of regulation of gene expression by two-component regulatory systems in bacteria?
A
- Sensor-transmitter protein monitors specific changes in the environment (e.g., level of nutrients, pH, solvent concentrations, osmolarity, etc.); spans the cell membrane with kinase on the inner part of the cell; kinase autophosphorylates upon receiving signal
- Response regulator protein obtains the phosphate from the kinase, causing change in conformation and activation of the effector domain
16
Q
Glutamine and arginine in DNA-binding proteins tend to make what kind of bonds with DNA?
A
• Hydrogen bonds with the nitrogenous bases of DNA
17
Q
What type of control is a repressor?
A
Negative control
18
Q
How does an auxiliary operator express control mechanisms?
A
additional repressor binding sites
19
Q
How does an effector express control mechanisms?
A
binding changes repressor conformation
20
Q
How does an activator express control mechanisms?
A
positive control
21
Q
How does an enhancer express control mechanisms?
A
binding sites for an activator
22
Q
How does a regulator express control mechanisms?
A
can express positive or negative regulation
23
Q
How does a two-component regulatory system express control mechanisms?
A
sensor-transmitter regulation
24
Q
How does DNA-bending express control mechanisms?
A
consequence of trans factor binding, can be +ve or -ve
25
How do conformational changes in RNAP express control mechanisms?
when promoter and regulatory binding sites are close or overlap, regulatory proteins can directly contact RNAP causing changes that promote formation of an open complex
26
How does DNA looping express control mechanisms?
direct contact can be achieved when even promoter and regulatory binding site are far apart
27
Define constitutive and regulated proteins. What is the difference between expression of constitutive and expression of regulated proteins?
* Constitutive proteins are always transcribed and translated
* Regulated proteins are only transcribed under certain conditions
28
What is the most important characteristic of binding sites for prokaryotic regulatory proteins (for example lac operon operator)? How are those binding sites different from the RNAP binding site (promoter)?
* Cis-element sequences are usually short inverted repeats
| * Promoters are asymmetrical (must position RNAP correctly)
29
Describe the most common structural motif found in a DNA binding domain of prokaryotic regulatory proteins.
* Helix-turn-helix motif: 20 amino acids long; 2 short alpha helices (7-9 amino acids) connected with a short ‘turn’
* Recognition helix and “stabilizing” helix form ~90° angle
* Each of the dimer’s helix-turn-helix motifs fit into one of the two neighbouring major grooves
* Also the N-terminal arms of the dimer’s monomers “wrap around” to the other face
30
Apart from the DNA binding domain, we have mentioned other two domains found in prokaryotic binding proteins. What are they?
* Effector binding domain – conformation of regulatory protein can be altered by small molecules
* Oligomerization (dimerization) domain
31
Thinking question: three adjacent genes are involved in arginine biosynthesis. (They are structural genes of arginine operon.) You have three DNA fragments, each containing coding sequence for one of these three structural genes, and you have used these fragments to make three different probes for northern blotting (one probe for each of the three genes).
You have isolated mRNA from bacteria and you have performed the agarose gel electrophoresis followed by northern blotting.
What result do you expect to get if you use each of these three probes as a probe in northern analysis? What would be the result if three genes do not make up an operon? You could use diagrams to explain your reasoning.
* If one probe – likely a polycistronic mRNA from active operon
* If three probes – not an operon
* If no probe – either wrong probe or operon is not active
32
Describe RNA Polymerase Structure
• Processive enzyme (50 nt/sec)
• Composed of six subunits:
o two α subunits – interaction with regulatory proteins (DNA control of initiation frequency)
o two large β subunits – catalytic subunits
o one ω subunit – required to restore denatured RNA polymerase in vitro to its fully functional form
o one σ70 subunit – interaction with promoter, initiation
• Holoenzyme – 6 subunits
• Core enzyme – without σ70 subunit
33
Discuss why are lac I- mutants trans-acting.
- I- is a constitutive mutations that results in structural genes always being expressed
- Trans acting since mutated repressor that cannot bind to the promoter, therefore con
34
Discuss positive and negative regulation of L-ara operon.
* The initiator region contains ara I, which codes for enzymes that act on arabinose
* ara C encodes the activator protein AraC
* When there is no arabinose, the AraC binds to ara O2 and ara I1 and bends DNA, hiding the promoter from RNA polymerase so enzymes are not synthesized
* Where arabinose is present, it binds to AraC, changing the conformation of the protein, so AraC binds to ara I1 and ara I2 making the promoter accessible to RNAP
35
Regarding the regulation of Trp operon, what do we call the amino acid tryptophan? Why?
* Tryptophan is co-repressor
* It is necessary to activate the aporepressor protein that then binds to the promoter to negatively regulate synthesis of the structural genes
36
What is meant by polycistronic mRNA? Give an example.
• mRNA that contains genes for more than one protein product (e.g., the lac operon)
37
What is catabolite repression? What is the role of Catabolite Activator Protein? Explain its action. (remember the # of operons it activates!)
* Catabolite repression – the negative control of the expression of enzymes involved in catabolism of energy sources other than glucose; the “glucose effect”
* The Catabolite Activator Protein (CAP) or cyclic AMP (cAMP) will induce or active the pathway for synthesis of lactose by complexing with RNAP when cell is starving from lack of glucose
* cAMP binds to CAP, which binds to CAP binding site upstream from RNAP binding site, stimulating transcription
* called pleiotropic or general activator
38
Define: repressor, co-repressor, aporepressor and inducer.
* Repressor – trans factors that bind to decrease the rate of transcription
* Co-repressor – activate aporepressors that are then capable of binding to decrease the rate of transcription
* Aporepressor – a repressor that cannot combine with an operator gene until it has combined with a corepressor
* Inducer – trans factors that inactive repressors thereby inducing transcription
39
Define effector and inducer. Give example
* Effector – small molecules that change the conformation of repressors by binding; either inducers or corepressors
* Inducer – trans factors that inactive repressors thereby inducing transcription
40
What are activators? What are enhancers?
* Enhancers – cis elements that, upon binding of an activator, recruits transcription factors that enhance the binding of RNA polymerase, thereby increasing the rate of transcription
* Activators – trans factors that act on enhancers
41
What is the role of auxiliary operators?
* Multiple operators (auxiliary operators) can increase or decrease the level of transcription dramatically (e.g., 20-fold to 500-fold with 2 other operators in lac operon)
* Can increase the local concentration of the repressor or cause DNA looping as repressor binds to many operators which prevents binding of RNA polymerase
42
Discuss the type of regulation of gene expression by two-component regulatory systems in bacteria?
* Sensor-transmitter protein monitors specific changes in the environment (e.g., level of nutrients, pH, solvent concentrations, osmolarity, etc.); spans the cell membrane with kinase on the inner part of the cell; kinase autophosphorylates upon receiving signal
* Response regulator protein obtains the phosphate from the kinase, causing change in conformation and activation of the effector domain
43
Glutamine and arginine in DNA-binding proteins tend to make what kind of bonds with DNA?
• Hydrogen bonds with the nitrogenous bases of DNA
44
What type of control is a repressor?
Negative control
45
How does an auxiliary operator express control mechanisms?
additional repressor binding sites
46
How does an effector express control mechanisms?
binding changes repressor conformation
47
How does an activator express control mechanisms?
positive control
48
How does an enhancer express control mechanisms?
binding sites for an activator
49
How does a regulator express control mechanisms?
can express positive or negative regulation
50
How does a two-component regulatory system express control mechanisms?
sensor-transmitter regulation
51
How does DNA-bending express control mechanisms?
consequence of trans factor binding, can be +ve or -ve
52
How do conformational changes in RNAP express control mechanisms?
when promoter and regulatory binding sites are close or overlap, regulatory proteins can directly contact RNAP causing changes that promote formation of an open complex
53
How does DNA looping express control mechanisms?
direct contact can be achieved when even promoter and regulatory binding site are far apart
54
Define constitutive and regulated proteins. What is the difference between expression of constitutive and expression of regulated proteins?
* Constitutive proteins are always transcribed and translated
* Regulated proteins are only transcribed under certain conditions
55
What is the most important characteristic of binding sites for prokaryotic regulatory proteins (for example lac operon operator)? How are those binding sites different from the RNAP binding site (promoter)?
* Cis-element sequences are usually short inverted repeats
| * Promoters are asymmetrical (must position RNAP correctly)
56
Describe the most common structural motif found in a DNA binding domain of prokaryotic regulatory proteins.
* Helix-turn-helix motif: 20 amino acids long; 2 short alpha helices (7-9 amino acids) connected with a short ‘turn’
* Recognition helix and “stabilizing” helix form ~90° angle
* Each of the dimer’s helix-turn-helix motifs fit into one of the two neighbouring major grooves
* Also the N-terminal arms of the dimer’s monomers “wrap around” to the other face
57
Apart from the DNA binding domain, we have mentioned other two domains found in prokaryotic binding proteins. What are they?
* Effector binding domain – conformation of regulatory protein can be altered by small molecules
* Oligomerization (dimerization) domain
58
Thinking question: three adjacent genes are involved in arginine biosynthesis. (They are structural genes of arginine operon.) You have three DNA fragments, each containing coding sequence for one of these three structural genes, and you have used these fragments to make three different probes for northern blotting (one probe for each of the three genes).
You have isolated mRNA from bacteria and you have performed the agarose gel electrophoresis followed by northern blotting.
What result do you expect to get if you use each of these three probes as a probe in northern analysis? What would be the result if three genes do not make up an operon? You could use diagrams to explain your reasoning.
* If one probe – likely a polycistronic mRNA from active operon
* If three probes – not an operon
* If no probe – either wrong probe or operon is not active
59
Describe RNA Polymerase Structure
• Processive enzyme (50 nt/sec)
• Composed of six subunits:
o two α subunits – interaction with regulatory proteins (DNA control of initiation frequency)
o two large β subunits – catalytic subunits
o one ω subunit – required to restore denatured RNA polymerase in vitro to its fully functional form
o one σ70 subunit – interaction with promoter, initiation
• Holoenzyme – 6 subunits
• Core enzyme – without σ70 subunit
