Histones and Transcription
Effect of histone deacetylation on gene transcription?
Makes the histone more positive, therefore, the histone binds the DNA more tightly
Less transcription of the gene
___ bases in DNA can be methylated without changing their base-pairing
Cytosine (C)
What is the function of:
HIV protease?
Reverse transcriptase?
Integrase?
HIV protease cleaves/processes the polyproteins that are made from the viral DNA/mRNA, this process allows the viral capsules to mature and become infectious
Reverse transcriptase, turns the viral RNA in the viral capsule into viral DNA that can be inserted into the genome
Integrase takes the viral DNA into the nucleus and inserts it in the host cell’s genome
What are the two essential components of any CRISPR editing system?
Cas9 protein
Guide RNA
What does a lysogenic viral life cycle look like?
A lysogenic virus is dependent on host cell transcription machinary, and therefore viral replication is dependent on transcription of the cell's healthy genome.
Life cycle includes a latent phase, and viral transcription doesn't occur until the cell is "activated"
acetylase / deacetylase
What is maintenance methylation?
Does it occur in prokaryotes, eukaryotes, or both?
When DNA is replicated. Only the template strand remains methylated. If replication was to occur again, without re-methyalting the other strand then the DNA would no longer be methylated / transcription may change.
However, and enzyme (methyl-transferase) does maintenance methylation and re-methylates the unmethylated strand after replication.
Hemi-methylated strands then turns back into a methylated strand
This occurs in BOTH prokaryotes and eukaryotes
In poliovirus, what is +-strand RNA? In poliovirus, what does RNA-directed RNA polymerase do?
+-strand RNA is the strand of RNA that is translated into proteins
RNA directed RNA polymerase makes a - template RNA strand with the +ssRNA. This template is then used to make more +ssRNA.
For bacteria, what is the main purpose of CRISPR?
The main purpose of CRISPR for bacteria is to identify viral DNA and cleave it, rendering it useless / unharmful.
How does cooperative binding help eukaryotic transcription respond to multiple signals?
Cooperative binding is when multiple protein transcription factors bind to a region of the DNA and together (with a combined effect) alter the structure of the DNA in such a way that the promoter sequence becomes stronger. The requirement of the multiple protein transcription factors, allows precise regulation of the promoter region.
Why is histone deacetylase a potential problem for gene therapy treatment?
Histone deacetylase does the opposite (makes histones more positively charged / increases binding strength with DNA). When foreign DNA is introduced to a cell it is often bound up with deacetylated histones as a defense mechanism to inhibit it’s transcription.
If the gene is not being transcribed, then it is likely NOT having it's intended therapeutic effect
What are CpG islands? AND are they always methylated?
Are all cytosine bases in DNA methylated?
CpG islands are CpG rich chromosome regions.
CpG is a cytosine base connected to a guanine by a phosphate (on the same strand) we include the P to differentiate from a CG base pair
80-90% of CpGs are methylated
Only around 1% of cytosine bases in DNA are methylated
Where is CD4 receptor found, and why is it important in the HIV life-cycle?
CD4 receptor is found on T-helper cell’s cell membrane. CD4 is a stimulator signal that the HIV virus uses to bind to / recognize T-helper cells and fuse with their cell membrane.
Briefly describe the two ways that CRISPR technology can be used to modify genes. What is its major advantage, versus using a retrovirus vector for gene therapy?
CRISPR technology can be used to cleave DNA at specific sequences. After the DNA strands are cleaved, the cell will repair the damage. If template DNA is provided, it can be incorporated via the cell’s natural homologous repair mechanism.
Therefore, CRISPR can be used to insert new DNA into a genome to fix mutated genes. Furthermore, CRISPR can also be used to mutate or silence specific genes, for research purposes.
The major advantage of CRISPR versus a retrovirus, is that CRISPR can cut specific DNA sequences and therefore, change the DNA / incorporate DNA at specific sites. While retroviruses incorporate DNA in more or less random locations within the genome.
CRISPR stands for?
Clustered regularly interspaced short palindromic sequences
How is a histone made "less" positive in the acetylase process?
Lysine residue is acetylated
An ammonium becomes and amine / deprotonated / loses a + charge
What is the purpose of methylation in prokaryotes?
For each of the following key terms, describe the similarity or difference between poliovirus vs HIV, and how those affect the courses of the diseases that they cause.
genetic material
Both are composed of +ssRNA
replication of genetic material
In HIV the +ssRNA is reverse transcribed in to ds DNA and then incorporated into the host genome for transcription when the T-helper is naturally activated
In poliovirus the +ssRNA is translated into proteins, one of those proteins is RNA directed RNA polymerase which replicates the +ssRNA forming a template (-ssRNA strand) from which more +ssRNA can be transcribed. Once the template strand is made, more viral RNA and viral proteins can be made. From the viral proteins and viral +ssRNA viral capsules are produced, which then enter into the bloodstream where they can infect other cells.
cell receptor latent/lytic
HIV requires the cell’s own transcription machinery, and therefore lies on the cell to naturally transcribe the viral DNA, and therefore, has a latent phase
Poliovirus is lytic, meaning that it does not require the cell’s machinery to produce more viral particles, it immediately starts reproducing the virus upon entry into the cell
In nature, in a CRISPR system are there two strands of RNA or one? Does this differ from engineered CRISPR systems.
In nature there are two RNA strands. One strand recruits Cas9 while the other functions as the guide strand.
In engineered CRISPR systems both strands have been combined into one strand for simplicity
Compare histone deacetylation / acetylation to DNA methylation in relation to transcription?
Histone deacetylation decreases transcription while histone acetylation increases transcription
While DNA methylation decreases transcription and induces histone deacetylation
How does the cell decide which histones to acetylate?
No one really knows :O
But... CpG methylation is involved
Is cytosine methylated before or after incorporation into DNA?
After
Describe the entire life cycle of HIV, from start to finish.
Costimulatory signals on the viral capsule and T-helper cell (CD4 and CCR5) initiate the viral capsule’s membrane and cell membrane (of the T-helper cell) to fuse.
Viral enzymes and viral RNA are released into the cell
Viral reverse transcriptase converts the viral RNA into viral DNA
Integrase carries the DNA into the nucleus of the cell and allows insertion of the viral HIV DNA into the host’s genome / chromosome.
The virus now “waits” for the infected T-helper cell to be naturally activated by the immune system
Upon activation the viral DNA is transcribed to mRNA
This mRNA serves two purposes: #1 produces new RNA viral genomes, and #2 produces the necessary proteins and enzymes for the viral particles
The viral particles form within the cell
Viral particles are released from the cell
Viral protease cleaves / processes the proteins that were produced, and the viral capsules become mature and infectious
The T helper cell eventually dies
What is the advantage of a CRISPR system vs. other genetic engineering systems? (Zinc finger nucleases, TALENs, ETC...)
CRISPR uses a single protein (Cas9) with an editable RNA segment. Therefore, it requires much less protein engineering AND is much easier to use.
What other DNA base can be methylated and transformed into another base?
Thymine when methylated is uracil, however, this methylation occurs before the base is incorporated.