Topic 1 Flashcards
Humans have __ pair(s) of autosomes and __ pair(s) of sex chromosomes
22 pairs of autosomes, 1 pair of sex chromosomes
What two sets of genomes do humans have?
The mitochondrial genome and the nuclear genome
How many mtDNA genes? Approximately how many nucleotides?
37 genes, approximately 16.6 kb
How many rRNA coding mtDNA genes?
2
How many tRNA coding mtDNA genes?
22
How many polypeptide coding mtDNA genes?
13
Approximately how many genes in nuclear chromosomes?
3.2 x 10^9
How do X and Y chromosomes differ?
- They are structurally distinct and are subject to different forms of genetics regulation.
- They also have their own patterns of inheritance (e.g. Y can only come from the father)
Females have 2 times more X-linked gene expression than males. How is this dosage imbalance corrected?
Through dosage compensation
- through inactivation of one of the X chromosomes in females.
What is the X chromosome inactivation theory?
In the somatic cells of normal females (but not males), one of the two X chromosomes is randomly inactivated early in development.
True or false: The inactivated X chromosome remains inactive for the rest of the female’s life
False
- The inactivated X chromosome becomes reactivated in oogenesis in the germ line
How was X chromosome inactivation initially identified?
X chromosome inactivation was initially identified through the discovery of a highly condense, heterochromatic structure called the Barr body in interphase cells.
Describe how X chromosomes are inactivated
On the X chromosome, there is a region called the X inactivating center (XIC). This region can be decorated with XIST RNA, which changes the morphology of the X chromosome and therefore decreases expression of genes in the X-linked gene.
How can scientists determine the function of XIST?
By doing a knockout with siRNA, CRISPR or site-directed mutagenesis. Can use Northern blots or qPCR to quantify RNA content after knockout.
What is Klinefelter syndrome?
- When a male has a 47, XXY trisomy.
- One of the X chromosomes will become inactivated like in females, but some genes on the “inactive” X chromosome escape X inactivation.
What are some of the symptoms of Klinefelter syndrome?
- infertility
- tall/thin with long legs (decreased muscle tone)
- hypogonadism (low testosterone)
- underdeveloped secondary sexual characteristics.
In Klinefelter syndrome, why do individuals with the genotypes 48 XXXY and 49 XXXXY have more severe phenotypes?
Because there’s more X leakage
Bacteriophage
Virus that infects bacteria
Lambda (λ) phage
DNA virus that infects the bacterial species, such as E.coli
How many bps in λ chromosome? How many genes?
~48,500 bp, ~50 genes
The λ coat contains how many proteins (from head to tail)?
~15
Prophage
A phage DNA (in its dormant state) that integrates into the bacterial chromosome
Lysogen
The bacterial cell that harbors the prophage
Lytic cycle
After bacteriophage injects DNA, DNA replicates and proteins are transcribed in bacteria, more bacteriophages produced in bacteria and then host lyses.
- kills host cell
Lysogenic cycle
Prophage is integrated into bacterial chromosome. Host is not lysed.
What happens if lysogen undergoes UV induction?
Lytic cycle is induced
On a plaque assay, if there’s more spots that means…
There are more bacteriophages
On a plaque assay, if there’s more clear plaques…
There is more lytic growth
On a plaque assay, if there’s more cloudy (turbid) plaques…
There is more lysogenic growth
If there is full growth on a plaque assay (no spots), this indicates that…
Cells are not being lysed
Explain the immunity of the λ-lysogen
Repressor molecules maintain the prophage in its dormant state. These repressor molecules prevent further λ infection and lysis.
- When the λ repressor protein (aka cI) is predominant, λ phage enters the lysogenic pathway (its DNA integrates into the host chromosome, and only the λ repressor itself is expressed)
Explain the effect of UV on the λ repressors
UV inactivates these repressors, so then another regulatory protein called Cro becomes predominant. When this protein is predominant, λ phage enters the lytic cycle (most of the λ genes are expressed, and viral replication/packaging ensue.
Cro protein general function
Control of repressor and other genes
cII protein
The cII protein is a transcription activator that enhances transcription of the λ repressor gene and hence stimulates production of the repressor protein (cI). When cII is abundant and active, infection proceeds through the lysogenic pathway.
Cro gene and cI (λ) gene status in lytic cycle
- Cro gene is on
- cI (λ) repressor gene is off
Cro gene and cI (λ) gene status in lysogenic cycle
- Cro gene is off
- cI (λ) repressor gene is on
Order of λ control regions (promoters) and genes from left to right on chromosome
PL, cI gene, PRM, PR, cro gene
In lytic cycle, describe the PL, PRM and PR promoter statuses
PL is on, PRM is off, PR is on.
- Results in no cI being expressed, but cro is expresssed (PR is cro promoter)
- N protein (gene that PL promotes) is also expressed.
In lysogenic cycle, describe the PL, PRM and PR promoter statuses
Only PRM is on, so cI is expressed but cro is not expressed
Operator general function
A DNA sequence that can be recognized and bound by a repressor protein to block transcription of the gene.
Promoter general function
DNA sequence that can be recognized and bound by an RNA polymerase to initiate transcription of the gene.
How many operators are there between the PRM and PR promoters?
3, from left to right: OR3, OR2, OR1
True or false: the RNA polymerase that transcribes the cro or cI gene in the bacteriophage comes from the virus
False. The RNA pol comes from the host.
The PRM promoter transcribes the ___ gene, and activator proteins (are/are not) required for transcription initiation
The PRM promoter transcribes the cI gene, and activator proteins are required for transcription initiation
Describe the structure of the cI (λ) repressor, in terms of amino acids and domains
- 236 amino acids in length (25,960 Da)
- 2 domains (N/amino domain and C/carboxyl domain)
- Attached by a 40 amino acid linker
The PR promoter transcribes the ___ gene, and activator proteins (are/are not) required for transcription initiation
The PR promoter transcribes the cro gene, and activator proteins are not required for transcription initiation
What are the 4 subdomains and their locations on the cI repressor?
C domain:
- tetramerization domain
- dimerization domain
N domain:
- activating region
- DNA binding domain
Can the λ repressor bind to the prophage operator as a monomer?
No; the repressor can associate with itself to form a homodimer. Only the dimers can cooperatively bind to the operators via their amino domains.
How many amino acids in Cro protein?
66
Cro monomers have (very high/very low) affinity for each other, which results in…
Cro monomers have very high affinity for each other and are almost always forming dimers
True or false: Cro binds the same operators as the cI repressor
True, but they play an opposing role because Cro promotes the lytic cycle instead.
What lab technique could you use study the binding interactions between the phage DNA and the DNA+ Cro protein?
- electrophoretic mobility shift assay (EMSA)
- basically just gel electrophoresis with two lanes, one lane is DNA and the other lane is DNA + protein. If protein bind to the DNA, then the fragment in this lane travels slower.
Which one of the following statements is false regarding the role of the λ repressor in regulation of phage transcription?
a) λ repressor bound to DNA excludes RNA polymerase
b) λ repressor cooperatively binds DNA
c) λ repressor lacks an activation domain
d) λ repressor recruits RNA polymerase to the promoter
c
If the cellular concentration of a repressor protein is several orders of magnitude greater than the dissociation constant for the repressor-operator complex, how often will the operator be bound by repressor?
The operator site will almost always be bound by repressor.
Describe what happens to the cI gene if the cI repressor binds at OR2
cI positively regulated the expression of its own gene by enhancing the binding of RNA polymerase to PRM
Describe what happens to the cro gene if the cI repressor binds at OR2
cI physically blocks RNA polymerase from binding the PR promoter and suppresses the transcription of the cro gene
- Is an example of the principle of exclusion, which is a very common mechanism of gene regulation
Describe what happens to the cI and cro genes when the cI repressor binds to OR3
- Negatively regulates the cI transcription by preventing RNA polymerase from binding at the PRM promoter
- No effect on PR: cro gene is on (lytic cycle activated)
Describe what happens to the cI and cro genes when the lambda repressor binds to OR1
- Negatively regulates the cro transcription by blocking RNA polymerase from binding to the PR promoter
- PRM is only weakly activated, because the repressor at OR1 is too far away to affect RNA polymerase
In the lysogenic state, >90% of the phages are found with repressors bound to OR1 and OR2 (but not OR3), while <10% of the phages are found with repressors bound to all three operators. What 2 factors affect this?
- Repressor dimer has an “intrinsic” affinity for each operator site (i.e., OR1 > OR2 > OR3)
- Interaction of repressor dimer at adjacent sites to facilitate OR binding (cooperativity from OR1/OR2 binding makes repressor lean away from repressor bound to OR2)
Given the choice, the repressor will bind to (this operator), but if the concentration of repressor increases, then the repressor will begin to bind at (this operator)
Given the choice, the repressor will bind to OR1, but if the concentration of repressor increases, then the repressor will begin to bind at OR2
True or false: When the cI repressor is bound to both OR1 and OR2, these dimers are in physical contact
True
Describe the cooperative binding involving the λ repressor
Cooperativity is seen as the binding of repressor at OR1 increases the affinity at OR2 for another repressor dimer
True or false: OR3 binding by the λ repressor is also strong
False
- Repressor binds to OR3 much more weakly than OR2 -> requires a much higher [repressor] for OR3 binding
Describe why the binding of the λ repressor to OR3 is weak
The repressor dimer at OR2 is unable to interact with a repressor at OR3 (to form a tetramer), because it is already “leaning” towards the dimer at OR1
True or false: RNA polymerase needs OR2 repressor bound to be recruited to PRM (only OR1 isn’t enough)
True
Describe how UV induces the lytic cycle
- UV causes DNA damage
- DNA damage activates the E.coli protein, RecA (a recombinase, promotes recombination during meiosis I and triggers DNA repair by activating protein degradation)
- RecA induces proteolysis of repressor monomers
- The split monomers cannot dimerize
Describe the SOS response in E.coli (6 steps)
- UV causes DNA damage
- Rec A is activated (an E.coli protein)
- RecA induces proteolysis of Lex A (transcription suppressor on promoter of UvrA, UmuC and UmuD)
- Transcription of UvrA, UmuC and UmuD is turned on
- When these genes are on, nucleotide excision repair is activated
- If nucleotide excision repair doesn’t work, the genes turn on even with the mutation (life or death choice)
How does proteolysis reduce [repressor]?
Decreased repressor dimer means decreased dimer bound at OR1 and OR2 so decreased repressor synthesis
True or false: Cro dimers bind cooperatively
False
- Cro dimers bind independently to OR sites
What lab technique can we use to examine when operator binding is maximized with varying [Cro]?
EMSA
What is the affinity of Cro for OR 1-3?
OR3>OR1=OR2
- Almost opposite of the λ repressor
When Cro is bound to OR3…
λ expression is turned off because the repressor prevents RNA pol from binding PRM
- Opens up PR to bind RNA pol to express Cro protein
λ repressor is said to be both a positive or a negative regulator, while Cro is said to be only a negative regulator. Why?
- λ negatively regulates its own expression when bound to OR3 (which barely ever happens). λ positively regulates its own expression when bound to OR1 and OR2 (it ENHANCES the binding of RNA pol to OR3). λ also negatively regulates Cro expression when bound to OR1 and/or OR2.
- When Cro binds to OR3, it negatively regulates λ expression (and does not enhance RNA binding to PR, just doesn’t block PR) but when it’s bound to OR2/OR1 or all OR sites, it negatively regulates its own expression
After the lambda repressor is degraded, the PRM promoter and OR sites are free of any repressor. What happens to PRM over time?
Eventually, the PRM is off.
What happens to cI and Cro gene expression when the lambda repressor binds to OR2 only (although this is rare)?
- Repressor physically blocks RNA polymerase from binding the Pr promoter and suppresses the transcription of the Cro gene
- cI gene still transcribed
Which gene is turned on when the lambda repressor is bound to OR3 only (even though this is rare due to cooperative binding)?
cI is off, Cro is on
What happens when the Cro protein binds to OR3 and OR2?
both cI and Cro are off
What happens when the Cro protein binds to OR3 and OR1?
both cI and Cro are off
Which of the following occurs more often in cells?
a) Cro binds OR3 and OR2
b) Cro binds OR3 and OR1
Both occur with equal probability because affinity of Cro for OR1 and OR2 are equal
PR is a strong promoter. What does this imply about its transcription?
It is always on unless repressed (constitutive)
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
True or false: The PRM and PR promoters can be occupied simultaneously
False
