Chapter 19 Flashcards
Yeast two hybrid system
Yeast Two-hybrid system
Protein A is fused to DNA-binding domain of Gal4. Protein B is fused to an activating domain.
Neither protein alone, when expressed in yeast cell, activates the reporter gene carrying Gal4- binding sites.
When both hybrid genes are expressed together in a yeast cell, the interaction between protein A and B generates a complete activator, and the reporter gene is expressed.
The Two-Hybrid assay can be used to screen a library of candidates (prey) that will interact with a known starting protein (bait)
DNA binding functions of activators
Activators have separate DNA-binding and activating functions
An example of eukaryotic activator Gal4. It activates the transcription of galactose genes for galactose metabolsim in the yeast.
Gal4 binds to 4 sites located 275bp upstream of the GAL1 gene. When bound there, in the presence of galactose, Gal4 activates transcription of the GAL1 gene 1000-fold.
Activating regions as not well defined structures
Activating regions are not well-defined structures
Instead of being characterized by structure, activating regions are grouped on the basis of amino acid content. They have been shown to form helical structures when interacting with their targets within the transcriptional machinery. They also have equally critical hydrophobic residues.
It is believed the a activating regions consist of reiterated small units, each of which has a weak activating capacity on its own. This is consistent with the idea that activating regions lack an overall structure and act simply as rather indiscriminate “sticky” surface. Imagine an activating region folds into precise three dimensional structure comparable to and DNA-binding domain, fragments of the structure would not be expected to retain the fraction of the DNA-binding activity of the intact domain.
1. The activating region of Gal4, is called an “acidic” activating region, reflecting a preponderance of acidic amino acid. (mutations that increase the overall acidity of the activating region will increase the activator’s potency.
2. SP1 activator has glutamine-rich activating regions.
3. CTF1 activator has proline-rich activating regions.
Activators recruit the transcriptional machinery to the gene.
Activators recruit the transcriptional machinery to the gene.
The eukaryotic transcriptional machinery contains numerous proteins in addition to RNA polymerase. Activators interact with one or more of these complexes and recruit them to the gene.
A typical acidic activating region can interact with components of the mediator and with subunits of TFIID.
How to define a region of DNA that recruit a specific protein?
ChIP assay (chromatin immunoprecipitation)
To identify any DNA than can bind to a given protein: ChI-chip or ChIP-seq
Activation of transcription through direct tethering of mediator to DNA.
Activation of transcription through direct tethering of mediator to DNA.
Similar activator bypass experiment works in yeast.
In this case, the GAL1 gene is activated, in the absence of the usual activator Gal4, by the fusion of DNA-binding domain of LexA to a component of the mediator complex (Gal11/Med15). Activation depends on LexA DNA- binding sites being inserted upstream of the gene.
Activators recruiting nucleosome modifiers to help the
transcriptional machinery bind at the promoter / initiate
transcription
Activators also recruit nucleosome modifiers that help the
transcriptional machinery bind at the promoter or initiate
transcription.
Nucleosome modifiers: (left) histone acetyltransferase (HATs), which add acetyl group. Addition of acetyl group to histone tails alters the interaction between those tails and adjacent nucleosomes. This modification is said to “loosen” chromatin structure, freeing up sites transcriptional machinery binding.
Acetylated nucleosomes also have higher affinity to TFIID (which is a different mechanism).
(right) SWI/SNF chromatin remodeling factors. Activator recruits a nucleosome remodeler, which alters the sturcture of nucleosomes around the promoter, rendering it accessible and capable of binding the transcriptional machinery.
Activators recruit additional factors needed for efficient initiation or elongation at some promoters
After phosphorylation by TFIIH of serine 5 in the “ tail”, polymerase initiates transcription. But in some promoters, it then pauses until a second phosphorylation on serine 2 is achieved through recruitment (by an activator) of kinase P-TEFb (which is found in the SEC complex.
• P-TEFb is a part of a larger complex, the SEC (super elongation complex), which releases paused Pol II from the proximal promoter. Pausing is mediated by the complex called NELF.
• A strong acidic activator like Gal4 is able to recruit P-TEFb/SEC along with the rest of the transcriptional machinery.
Transcriptional repressors
Transcriptional repressors
• In bacteria, repressors can bind to the sites overlapping with the promoters, and block RNA polymerase from binding there. They can also bind to the sites adjacent to the promoters, and by interacting with polymerase bound there, and inhibiting the enzyme from initiating transcription. They can also interfere with the action of activators.
• In eukaryotes, we see all of these except the first one. We also see another form of repression, perhaps the most common in eukaryotes: repressors can recruit nucleosome modifiers. In this case, the enzymes have the opposite effects to those recruited by activators. The compact the chromatin or remove groups recognized by the transcriptional machinery.
• Example, histone deacetylase represses transcription by removing acetyl groups from the tails of histones. (not absolute, histone deacetylase is also recruited to active genes to ensure transcription fidelity. Nucleosome are deacetylated behind elongating Pol II to prevent the use of “cryptic” promoters within the transcription unit.)
• Other enzymes add methyl groups to histone tails, and this frequently represses transcription, although in some cases it is associated with an actively transcribed gene.
Enhancer mechs
Mechanism enhancer action is still a mystery. Cohesin protein has been implicated in stabilizing enhancer-promoter loops. A protein called Chip aids in communication between enhancer and promoter. (Chip mutation in Drosophila affects the strength of enhancer activity of an enhancer on cut gene)
Four models:
1.Change in topology. Binding of a gene-specific transcription factor causes supercoiling, facilitating the binding of general transcription factor and polymerase to the promoter.
2.Sliding. A transcription factor binds to the enhancer and slides down the DNA to the promoter.
3.Looping. A transcription factor binds to the enhancer, and by looping out the DNA in between, binds to and facilitates the binding of general transcription factors and polymerase to the promoter.
4.Facilitated tracking. A transcription factor binds to the enhancer causes a short DNA fragment to loop out downstream. Increasing size of the loop allows the factor to track along the DNA until it reaches the promoter.
Insulators blocking activation of enhancers
Insulator blocks the communication between the enhancer and the promoter. Insulators often bind a large zinc-finger protein CTCF. It is now believed that CTCF also binds cohesin and this complex forms a chromosomal loop with the nearest promoter, thereby precluding enhancers distal to the insulator for forming a similar loop.
Insulators also inhibit the spread of chromatin modifications. Propagation of certain repressing histone modification over stretches of chromatin (multiple genes) lies at the heart of a phenomenon called transcriptional silencing.
Experimental implication: A transgene inserted at random into mammalian genome is often “silenced” because it is incorporated into the dense form of chromatin called heterochromatin. If insulators are placed upstream and downstream from that gene, they protect it from silencing.
Locus control regions and beta globin gene
Appropriate regulation of some groups of genes requires locus control regions
The human globin genes are expressed in red blood cells of adults and in various cells in the lineage that forms red blood cells during development. Different globin genes are expressed at different developmental stages: start with ε (in the fetus), then the γ gene, followed by δ and culminating with the expression of β-globin after birth.
The β-globin gene (which is expressed in adult bone marrow) has two enhancers: one upstream of the promoter and the other downstream. Only in adult bone marrow are the correct regulators are active and present in appropriate concentrations to bind thee enhancers. But more than this is required to switch on the various globin genes in the correct order.
Regulation by LCRs
Regulation by LCRs
Locus control region, or LCR, is found 30- 50kb upstream of the whole cluster of globin genes. A similar GCR (global control region) controls HoxD gene cluster.
The LCR is made up of multiple sequence elements. Some have property of enhancers. Other parts of the LCR act more like insulator elements, and still others seem to have properties of promoters.
Recent experiments using chromosome conformation capture technique allows the locations of LCR and promoter to be visualized in cells during activation. Regulatory proteins that bound to the upstream regulatory sequences are found in close proximity to the promoters as that promoter is active. This is consistent with the idea that proteins bound at LCRs interact with others at the promoter, with the intervening DNA looping out to accommodate the interaction.
Activators work synergistically to integrate signals
When multiple activators work together, they often do so synergistically. That is, two activators working together is greater than the sum of each of them working alone.
• Synergy can result from
(A) Cooperative binding through direct
interaction between two proteins;
(B) Similar effect is achieved by both protein interacting with a common third protein;
(C) The first protein recruits a nucleosome remodeler whose action reveals a binding site for the second protein;
(D) The binding of the first protein unwind the DNA from the nucleosome a little, reveals the binding site for the second protein.
Co binational control and its different levels
Combinatorial control lies at the heart of the complexity and diversity of eukaryotes
(a) Gene A is controlled by 4 signals.
(b) GeneBiscontrolledby3 signals.
Each signal is communicated to a gene by one regulatory protein. Regulatory protein 3 acts at both genes, in combination with different additional regulators in the two cases.
Two levels of combinatorial control:
A. Multiple activator work synergistically
B. A regulator controls multiple genes
Transcriptional repressors
Transcriptional repressors
• In bacteria, repressors can bind to the sites overlapping with the promoters, and block RNA polymerase from binding there. They can also bind to the sites adjacent to the promoters, and by interacting with polymerase bound there, and inhibiting the enzyme from initiating transcription. They can also interfere with the action of activators.
• In eukaryotes, we see all of these except the first one. We also see another form of repression, perhaps the most common in eukaryotes: repressors can recruit nucleosome modifiers. In this case, the enzymes have the opposite effects to those recruited by activators. The compact the chromatin or remove groups recognized by the transcriptional machinery.
• Example, histone deacetylase represses transcription by removing acetyl groups from the tails of histones. (not absolute, histone deacetylase is also recruited to active genes to ensure transcription fidelity. Nucleosome are deacetylated behind elongating Pol II to prevent the use of “cryptic” promoters within the transcription unit.)
• Other enzymes add methyl groups to histone tails, and this frequently represses transcription, although in some cases it is associated with an actively transcribed gene.
