Topic 1 (Genetic switch) Flashcards
True/False? The X and Y chromosomes have the same genetic regulation mechanisms
False. Females have two X chromosomes while males have one. To counteract overexpression of X in females, one X chromosome is condensed into a Barr body
Describe the formation of a Barr body
X-ist RNA binds to the XIC (X inactivating center) on the X chromosome. This condenses the chromosome into heterochromatin
True/False? X chromosomes are randomly selected for inactivation
True
When an X chromosome is condensed into a Barr body, are all of its genes completely silenced? Explain
No. About 85% of the genes are silenced. Mechanism is not covered
List the ways a researcher may test for the presence of an mRNA strand ***I don’t actually know
Northern blot
RT-qPCR
Gel electrophoresis
List the ways a researcher may test for the presence of a DNA strand ***I don’t actually know
Southern blot
Gel electrophoresis
qPCR
List the ways a researcher may test for the presence of a protein ***I don’t actually know
Western blot
SDS-PAGE
Gel electrophoresis
What is a bacteriophage? Give a course relevant example
A virus that infects bacteria. Lambda phage
What is a prophage? Which bacteriophage cycle is it involved with?
The dormant state of a phage that has been integrated into DNA. Important for the lysogenic cycle
What is a lysogen?
An infected bacterial cell that has a prophage integrated into its genome
Describe the basic steps of the lytic cycle
Bacteriophage attacks cell and hijacks replication machinery (RNA polymerase) to make progeny (baby bacteriophages). Once enough progeny have been made, the cell bursts open, releasing the new bacteriophages into the surrounding environment
Describe the basic steps of the lysogenic cycle
Bacteriophage attacks cell and uses integrase to integrate its DNA into the bacterial genome (becomes a prophage). Stays dormant until some form of DNA damage (UV) catalyzes the conversion of the lysogenic cycle into the lytic cycle
A clear plaque results from a plaque assay. Explain. What can you infer about the phage’s potency?
Bacteriophages are in the lytic cycle. Bacterial cells are killed, so no cloudiness is present. Lethal potency
A cloudy plaque with some clear spots results from a plaque assay. Explain
Bacteriophages are in the lysogenic cycle. Cloudiness indicates live bacteria while clear spots indicate lysed bacteria.
What is the role of lambda repressors for the continuation of the lysogenic cycle?
They maintain the prophage in the dormant state and prevent further phage infection and subsequent lysis by sequestering phages entering the cell
What is the role of UV in switching the state of the phage cycle?
Switches from lysogenic to lytic by inactivating lambda repressors. This allows the expression of the Cro gene
What protein is transcribed when PL is active? Which cycle is this associated with?
N protein. Lytic
What protein is transcribed when PR is active? Which cycle is this associated with?
Cro protein. Lytic
What protein is transcribed when PRM is active? Which cycle is this associated with?
Lambda protein (cI). Lysogenic
Which operators control PR?
OR2, OR1
Which operators control PRM?
OR3, OR2
Where does the RNA polymerase come from that transcribes the lambda prophage?
The host
True/False? PR and PRM require activator proteins for transcription
False. PRM is considered a weak promoter and requires activator proteins. PR is considered a strong promotor and does not require activator proteins
PR is a strong promoter. What does this imply about its transcription?
It is always on unless repressed (constitutive)
Which two domains does the lambda repressor N domain contain?
Trans-activating domain and DNA binding domain
Which two domains does the lambda repressor C domain contain?
Dimerization and tetramerization domain
Truse/False? Lambda repressor dimerization is cooperative
False. Its the dimers themselves that cooperatively bind to the operators
What is an electrophoretic mobility shift assay (EMSA)?
A type of gel electrophoresis where both DNA and DNA-protein complexes may be compared
True/False? Cro dimers bind to the same operators as the lambda repressors, but have opposing effects on transcription
True
Describe how the lambda repressor positively regulates its own transcription
When a cI dimer is bound to OR2, it enhances the ability of RNA polymerase to bind to PRM for the transcription of the cI gene
Describe how the lambda repressor negatively regulates Cro gene transcription
When a cI dimer is bound to OR2, it physically prevents RNA polymerase from binding to PR for Cro gene transcription
Describe how the lambda repressor negatively regulates its own transcription
When a cI dimer is bound to OR3, it prevents RNA polymerase from binding to PRM for cI gene transcription
Describe how the lambda repressor negatively regulates Cro gene transcription and its own transcription
When a cI dimer is bound to OR1, RNA polymerase cannot bind to PR for Cro gene transcription. RNA polymerase cannot bind to PRM because the dimer is bound at a site too far away to be effective as an enhancer
List the relative affinities OR1, OR2, and OR3 have for the lambda repressor. What effect does this have on gene transcription?
OR1>OR2>OR3. This means that 90% of the time, cI is bound to both OR1 and OR2, increasing cI transcription and inhibiting Cro transcription
True/False? A cI dimer bound to OR1 increases the intrinsic affinity of OR2 for cI. If true, what is this called?
True. Cooperative binding
What gene is turned on when a cI dimer is bound to OR1?
Neither, both Cro and cI are off
What gene is turned on when a cI tetramer is bound to OR1 and OR2?
cI is on, Cro is off
What gene is turned on when a cI dimer is bound to OR3?
Cro is on, cI is off
What gene is turned on when a cI dimer each is bound to OR1, OR2, and OR3?
Neither, both Cro and cI are off
True/False? A cI tetramer bound to both OR1 and OR2 increases affinity for OR3. If true, what is this called?
False. When cI tetramerizes, the dimers change conformation. The dimer at OR2 “leans” towards the dimer at OR1, preventing any further interactions with OR3
True/False? Once dimerized, lambda repressor proteins cannot disassociate from one another
False. They are constantly disassociating and reassociating
Describe the function of RecA
It acts as a recombinase during meiosis I, triggers DNA repair mechanisms when activated by UV, and activates repressor monomer proteolysis during DNA repair
RecA lowers the amount of repressor monomers in the cell. What effect does this have on cI transcription and the lysogenic cycle?
low [repressor monomer] = low [repressor dimer] = low [repressor dimer bound at OR1 and OR2] = low repressor transcription = decrease in lysogenic cycle
True/False? Like cI, Cro dimers bind cooperatively to the OR sites
False, they bind independently
List the relative affinities OR1, OR2, and OR3 have for Cro
OR3>OR1=OR2
Is Cro a positive or negative regulator?
Cro is strictly a negative regulator
Describe how the Cro protein negatively regulates cI gene transcription
When Cro is bound to OR3, RNA polymerase is unable to bind to PRM, so cI transcription is inhibited
What methods can be used to study Cro cooperative/independent interactions with DNA?
EMSA with varying levels of [Cro]
What gene is turned on when a Cro dimer is bound to OR3?
Cro is on, cI is off
Describe how the Cro protein negatively regulates cI gene transcription and its own gene transcription. Is there another combination of operators that can produce the same effect? Is one way more favourable than the other?
When Cro is bound to OR3 and OR2, or OR3 and OR1, or all three, RNA pol cannot bind to PRM or PR, so transcription for both genes is turned off. Either possibility has an equal probability of occurring because Cro binds to OR2 and OR1 with equal affinity
What gene is turned on when a Cro dimer each is bound to OR3 and OR2?
Both off
What gene is turned on when a Cro dimer each is bound to OR3 and OR1?
Both off
What gene is turned on when a Cro dimer each is bound to OR3, OR2, and OR1?
Both off
Which of the following statements is true regarding cooperative binding?
a) it decreases the concentration of protein required to fill adjacent protein binding sites on DNA
b) it increases the concentration of protein required to fill adjacent protein binding sites on DNA
c) it decreases the affinity of a protein for its correct binding site on DNA
d) it is observed only in DNA-binding proteins
A. Cooperative binding refers to the increase in affinity a protein has after binding to another protein, its ligand, etc
A lambda phage lysogen is largely immune to lysis by lambda phages introduced into the cell later. Why?
a) the lambda prophage produces Cro, which will bind to any lambda phages entering the cell and prevent induction of the lytic pathway
b) the lambda prophage produces cI and cII proteins, which will bind to any lambda phages entering the cell and prevent induction of the lytic pathway
c) the lambda prophage produces cI and cII proteins, which block phage attachment to the cell and prevent induction of the lytic pathway
B. cI and cII prevent lysis by disabling any phages that have entered the cell (lysogen)
The presence of lambda repressor in an infected E. coli cell most directly requires:
a) transcription from the PR
b) transcription from the PL
c) translation of Cro
d) transcription from the PRM
D.
