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Flashcards in Lecture 2 Deck (14)

What are some of the gene regulatory proteins that assemble together as complexes on DNA?

GAPs and a coactivator or repressors and corepressors


What is combinatorial control?

The multiple proteins binding to multiple sites that work together to initiate transcription. This bunch of different transcription factors determine the rate at which any given gene will be transcribed.


How does the position of enhancers in DNA affect binding of transcription factors?

1. motif orientation: if one site is not palindromic the wrong orientation will prevent cooperative binding
2. motif relative position: e.g. three sites need to be in the right order in order for cooperative binding to occur
3. motif distance: helical phasing. If sites are 10 bases apart (20, 30 etc) they are separated by complete helical turns and are able to interact. However TFs separated by 5/15/15bp etc are on opposite faces of the helix and are therefore unable to bind cooperatively


How can suites of genes be coordinated to be turned on at one particular point in time?

Through combinatorial control: one protein can coordinate the expression of several different genes e.g. a glucocorticoid receptor becomes activated in the presence of the glucocorticoid hormone and once activated is able to bind to a number of proteins already present and activate transcription of a number of different genes.


What are some ways in which the activity of a gene regulatory protein can be regulated?

1. protein synthesis (of course the protein needs to be synthesised in the first place in order to act)
2. Ligand binding (e.g. the glucocorticoid hormone)
3. covalent modification (e.g. addition of a phosphate group)
4. addition of a second subunit
5. unmasking (e.g. an inhibitor needs to be phosphorylated in order to be removed from the protein of interest)
6. stimulation of nuclear entry (needs to be able to enter the nucleus to bind DNA)
7. release from membrane (there is a subset of transcriptional regulatory proteins that are membrane tethered and need to be cleaved so that a region of that protein can move away from the cell membrane and enter the nucleus


Give an example of a single gene regulatory protein triggering the formation of an entire organ.

In drosophila, expression of eyeless in the eye precursor cells leads to eventual activation of 2000 genes. In a fly with Ey gene artifically expressed in leg precursor cells you get formation of an eye on the leg, thus demonstrating its influence. In drosophila with no Eyeless, eyes are not formed.


How can the addition or removal of ligands affect gene expression?

1. addition of ligand to bound repressor protein removes it and thus switches on gene
2. removing a ligand from a bound repressor protein switches gene off by removing repressor protein
3. addition of ligand to bound activator protein switches gene off by removing it from dna
4. removal of ligand from bound activator protein switches gene off by removing it


Describe a simple genetic switch.

The E.coli lac operon:
- lacZ gene is regulated by an activator (CAP) and the Lac repressor
- lacZ produces B-galactosidase (breaks down lactose into galactose and glucose)
- needed ON when lactose is high and glucose is low
- t/f in high glucose and lactose OFF > CAP not bound
- high glucose and low lactose > repressor protein bound to operator site
- both low > Activator binds to DNA but repressor still present so gene is off
- low glucose/high lactose > gene on
- high lactose binds to the Lac repressor and removes it from DNA
- high glucose decreases the amount of cAMP and loss of cAMP fro the CAP activator causes it to dissociate from DNA


Describe in basic terms the control region of the globin genes in eukaryotes.

Beta-globin is the main globin protein used in adults. It has three exons and two introns. There are gene control regions both upstream and downstream of the gene, each with many binding sites and regulatory proteins.
However the Beta- globin gene is located in a cluster of globin genes.
All globin genes are exclusively transcribed in erythroid cells, but at different stages of development. E-globin in the early embryo, gamma-globin in the later embryo and fetus, delta and beta globins in the adult (each has different oxygen binding capacities.)
Each has a set of regulatory proteins to turn the gene on at the appropriate time.
They also share a common regulatory region far upstream (the Locus Control Region) that amplifies transcription to cause the blood cell precursors to make massive amounts of globin.


Give an example of a tissue-specific transcription factor.

Experimental induction of MyoD in fibroblasts can cause them to express muscle-specific genes (specific actins, myosin, tropomyosin, troponin) and fuse into multinucleate muscle-like cells.
Therefore this single protein/gene is able to trigger a cascade that leads to formation of a new tissue type.


What is one way in which transcriptional repressors are important?

Can be required to maintain a cell state by keeping specific genes silent.
Rudolf Jaenisch Lab 2006 Nature:
- analysis of genes repressed by the Polycomb complex in mouse ES cells
- Found that 512 genes were specifically repressed by this complex and were mainly developmental regulators
Conclusion: genes that are expressed in stem cells are those required for cell proliferation and "housekeeping"
Differentiation and developmental genes must be kept silent.


How was the stripe 2 enhancer experimentally worked out?

Created lots of transgenic animals where they put pieces of DNA from the Eve promoter upstream of the lacZ, each with different deletions. By making observations of LacZ expression scientists were able to determine the key region of the promoter required for LacZ expression.
Secondly, by mutating expression of Giant or the Giant binding site you can observe expansion of expression of stripe 2 anteriorly.


How do patterns of gene expression become fixed? How does a differentiated cell prevent accidental expression of inappropriate genes?

Many genes are inactivated via DNA methylation. E.g. methylation of promoter region of gamma-globin gene prevents binding of transcription factors. Covalent methylation.
Histones can also be methylated: methylation of histone tails condense nucleosomes and thus represses transcription. Acetylation uncondenses nucleosomes and allows access to RNA polymerase and transcription factors.
The polycomb protein binds to trimethylated histone 3 Lysine 27 (H3Me3K27). Polycomb is part of a group of proteins that act together as the Polycomb Repressor Complex (PRC1) to repress transcription.


What is a reporter gene?

A gene that can be used experimentally (transgenic) to show the activity of an enhancer.