Final 21 Flashcards
What type of regulation is dominated in eukaryotes?
Positive regulation.
What is special about eukaryotic gene expression?
Access to euk promoters is hindered by chromatin structure.
What 5 things will you see on a typical regulatory sequence of a gene in euk?
Hsp 70: Core Promoter and the Proximal Promoter
Enhancers/Silencers
Insulators
Loci
What is the mediator complex?
It is a co-transcriptional activator that interacts with TF and RNAP. It relays information from a distal regulatory elements to RNAP, acting as a molecular bridge.
Whar are co-activators?
Proteins that increase gene expression by binding to another protein or activator, NOT DNA itself.
What are two types of chromatin alterations>
Modification and Remodeling.
What is chromatin modification?
The addition/removal of modifications on histones.
Requires ATP.
What is chromatin remodeling?
The movement of nucleosomes around DNA.
Requires ATP.
What are the 4 domains of a typically transcription factor?
DNA Binding Module
Dimerization Module
Activation Module
Regulatory Module
Activators can do three things, what are they?
- Recruit transcriptional machinery to the gene.
- Recruit nucleosome modifers.
- Recruit additional transcription factors needed for effective initiation/elongation
RNAPII Binding Requires 5 Types of Proteins, what are they?
- Transcription Activators (Enhancers(
- Architectural Proteins to Facilitate DNA Looping
- Chromatin Modification/Remodeling Proteins
- Co-Activators/Co-Repressors
- Basal Transcription Factors
What is the general choreography of transcription activation in 3 steps?
- Gene activator proteins bind to chromatin and recruit the chromatin remodeling complex to alter the DNA in a more open complex.
- Recruitment of other activator proteins along with the mediator complex.
- Basal transcription factors come in.
What two ways are DNA packaged?
Heterochromatin and Euchromatin
What is heterochromatin and Euchromatin DNA? Give three distinct differences between each.
Heterochromatin:
Densely packed/Transcriptionally Inactive
DNA sequence is repetitive
Histone 3-Lysine 9 is Methylated
Euchromatin:
Less Condense/Transcriptionally Active
DNA sequence is unique
Histone 3-Lysine 4 is Methylated
What has to happen to heterochromatin before you get transcription?
You have to get anti-repression first, before you get true activation.
Anti-repression will make your heterochromatic DNA turn into euchromatic DNA.
How does remodeling a chromatin help to activate a gene?
Remodeling will uncover the DNA binding site, otherwise, it would not be accessible due to its tight compaction.
How do modifications help activate a gene?
It can create specific binding sites for proteins or it can open/unravel the chromatin (via acetylation)
What does acetylation do to chromatin?
Acetylation induces a conformation change in core histone interactions with DNA and it recruits bromodomains.
Example how acetylation makes chromatin less packed.
Lysine-rich tails (positively charged) on the histones bind tightly to negatively-changed DNA, blocking access to the promoter sequence.
Addition of an acetyl-group will negate those positive charges on Lysine, disrupting the interaction between DNA and chromatin.
What are two things that acetylation does?
- Unravel DNA 2. Serve as a marker for other proteins (Bromodomains)
Are chromatin remodeling complexes dependent on ATP?
Yes.
A remodeler can bind to the chromatin or to an activator that is stuck on DNA and through ATP hydrolysis, it can move around the nucleosomes to unravel DNA.
Explain successive histone modification during transcription initiation.`
When a cell is being attached, interferons (cytokines) will be secreted.
In response to the interferon, an activator protein is going to bind to chromatin and recruit hitstone acetyltransferase, where it will acetylate Histone H3-L9. This will recruit a modifying enzyme that will phosporylate HH3-10. Where another modifying enzyme H3K14.
Once this has happened, we have set up the histone code to recruit the histone remodeling complex, which recognizes these modified lysines and serines.
What are three ways in which eukaryotic repressors can operate?
- Competitive Binding - repressor competes with the activator in binding to its site
- Masking the Activation Surface- both the activator and repressor are bound, but the repressor is inhibiting the functional active part of the activator
- Direct interaction with general transcription factor - repressor is inhibiting a transcription factor directly
What is true about chromatin remodelers and modifiers?
That they can act in the different direction.
i.e. histone de-acetylase
Explain the Gal4P pathway. What is it used for?
GAL4P recruits RNAP through a co-activator complex.
GAL4P is an activator that is always bound to DNA and its role is to recruit the chromatin remodeler. Its function is inhibited by GAL80P, which is bound to GAP4).
Which exposed to galactose, the gene is going to want to turn on to metabolize galactose. When GAL3P is complexed with galactose, it will interact with GAL80P, dissociating it, and allowing for GAL4P to function.
SAGA then gets recruited by GAL4P and now the chromatin can be remodeled.
So pretty much GAL80P was inhibiting GAL4P’s fuction..
What is combinational control?
Basic Principle: binding of one activator helps the binding of the secondary activator.
What are four examples of combinational control/cooperativity/synergy?
- A & B have to recruited together or one can be recruited first, which will recruit the other.
- Two TF ned to associate with the binding site before a co-activator can be recruited.
- Binding of one TF will allow for the recruitment of a chromatin remodeler, opening up a site for another TF.
- Binding of one TF induces a conformational change, opening up a site for another TF.
Explain interferon B and NFKB.
Inteferon B -> signal that needs to get produced with the help of NFKB, a transcription factor that needs to go nuclear but IKB is inhibiting the NLS signal of NFKB.
When the IKB gets phosphorylated, it will be set up for ubiquitination, leading to degradation.
The NLS signal will now become available and it can go into te nucleus.
Cooperative binding of NFKB. Explain.
Once NFKB is in the nucleus, many other TF are going to have to help to initiate transcription.
Normally, the gene that codes the the interferon is in a kinked conformation.
To activate this gene, you must first de-kink it, so a protein called HMGA-1 will bind to DNA, allowing it to open up the sequence.
Now all of transcription factors along with NFKB can bind an transcription can occur.
What is transcriptional silencing?
It is where large chromosomal regions are not transcribed for long periods of time. The chromatin structure is said to be heterochromatic.
What are SIR proteins and what do they do?
SIR proteins help to maintain the heterochromatic formation of a gene, silencing it.
What is the most important SIR protein and why?
SIR-2 because it have acetyl de-acetylase function.
Describe gene silencing by modifying histones.
- SIR proteins (1, 2, 3, and 4) are recruited by proteins already bound on DNA (Orc1, Rap1, Abf1).
SIR 3 de-acetylases lysines on the histones, recruiting more SIR proteins to the site, leading to more modifications.
With the help of all of the SIR proteins along withe the enzymatic activity of SIR 2, the you condensing of the chromatin, rsulting in gene silencing.
How can DNA methylation be involved in gene silencing? Where does it methylate?
MeCP2 binds to methylated DNA, recruiting Sin3A, a transcriptional co-repressor that has histone de-acetylase activity.
The cytosine on CpG islands are often methylated. This methylation laters the DNA and acts as a signal.
Methylated binding proteins, MeCP2 bind to these methylated cytosines and recruit the chromatin-remodeling complex, Sin3A.
Sin3A has de-acetylaton activity, causing the chromatin to condense, resulting in gene silencing.
What are insulators?
sequences to which proteins can bind to an by binding, they can prevent activator proteins fro affecting certain areas of the genome.
Explain the insulator example in class.
In the maternal chromosome, the insulator binding region is bound by a protein called CTFT. This silences the expression of Igf-2, but allows for H19 to be expressed.
in the paternal chromosome, the insulator isn’t present, therefore, methylation occurred over the ICR region and to the H19 region since it is hearby. This will cause silencing of the H19, and allow for Igf2 to be expressed.
How does spreading of the gene silencing mark be controlled?
Silencing can spread, but there are boundary elements that will contain this spread.
Boundary elements prevent spreading of silencing by inducing a these loops that will associate with the nuclear envelope. The silencing mark will be unable to jump from loop to loop.
