Transcriptional regulation - Eukaryotes (19) Flashcards
Differences between prokaryotes and eukaryotes
- Naked DNA in prokaryotes versus chromatin in eukaryotes (extra layer of regulation)
- Different upstream regulatory sequences
How is chromatin a barrier to transcription in vitro?
In eukaryotic cells the DNA is confined within the nucleus, wrapped around octamers of histone proteins (nucleosomes) to form the chromatin. The chromatin looks like beads of nucleosomes on a string of DNA. There is approx. 147 bp of DNA in a nucleosome and 80 bp of linker DNA.
When DNA is wrapped around in nucleosomes, the DNA regulatory sequences are not accessible to the basal transcription machinery. When researchers tried to transcribe chromatinized DNA template, there was no RNA synthesis despite having all the necessary partners present (RNAP, rTNPs, GTFs, template DNA). This demonstrated how chromatin acted as a barrier to transcription.
Why and how can chromatin be closed or opened?
CLOSED: Nucleosomes are very close to each other and don’t allow transcription factors, regulators or RNAP to access chromatin sequences -> Regulators & basal transcription machinery can’t access DNA sequences -> Gene OFF
OPEN: —II— can access DNA sequences -> Gene ON
The chromatin can’t be closed all the time, because then there would be no transcription. Therefore, there are mechanisms in eukaryotes that will keep the chromatin opened or closed depending on signals present. In this way, rather than only being a barrier, the chromatin functions as an extra layer of regulation -> Advantage over prokaryotes.
The opening happens in a regulated manner via
- Displacement (moving out of the way) or eviction (removal) of nucleosomes performed by chromatin remodeling enzymes
- Histone modifications (e.g. Acetylation) ->Trigger relaxation of chromatin structure
Regulators
Proteins that bind to specific DNA regulatory sequences outside promoters to regulate transcription
What are the differences in regulatory sequences in prokaryotes vs eukaryotes?
Regulatory sequences are generally found upstream of the core promoter.
Simple organisms generally have simple signals to implement and don’t need that many regulatory sequences. The more complex the organism, the more complex and numerous are the regulators and regulatory sequences because of the need to integrate more complicated signals.
- Bacteria have a promoter and some regulatory sequences
- Yeast (simple eukaryote) start having some more of the upstream regulatory sequences
- Humans (complex eukaryote) have tons of different types of regulatory sequences that can be located upstream, downstream, and also very far away from these TSS/core promoter (enhancers)
Same principals but increasing complexity of regulation from prokaryotes to mammals
Activators -> Bind to regulatory sequence -> Recruit RNAP -> Transcription
Repressor -> Bind to regulatory sequence -> Prevent recruitment -> No transcription
Eukaryotic regulation can be extremely complex compared to prokaryotes:
- Nature and number of signals to integrate requires more regulators
- Regulators can act at multiple levels: DNA, nucleosomes, regulatory sequences, regulatory proteins, etc.
- Coactivators
Difference in regulation of the 3 RNA polymerases
RNAP I and III:
- Few highly transcribed genes and few regulatory mechanisms
RNAP II:
- Majority of transcribed genes (protein-coding genes)
- Large variation of transcription rates
- Numerous regulatory mechanisms
Separated functions in eukaryotic activators: DNA-binding and activation (example: yeast activator gal4)
DNA binding and activation functions are separated:
- A DNA-binding domain (DBD) performs DNA binding
- An activation domain (AD) performs activation of transcription
Gal4 is used as a model for eukaryotic activators, and is responsible for transcription of GAL genes, including GAL, in response to galactose.
In the presence of galactose:
- Binding of Gal4 DBD to 4 DNA-binding sites in UASg (Upstream Activating Sequence for GAL genes)
- 1000x activation of GAL genes transcription including GAL1
- GAL genes encode the enzymes involved in galactose metabolism
(not very different from CAP activator)
The experiments that revealed functional separation between DBD and AD
The reporter assay & The domain swap assay
The reporter assay
Simplest assay you can do to study transcriptional regulation. Is the protein an activator or repressor of transcription? Is it working in transcription whatsoever?
Based on a construction in which you will have a promoter region and a reporter gene (e.g. Luciferase, GFP - green fluorescent protein, or LacZ).
- The DNA-binding site of an activator (e.g. UASg for Gal4) is placed upstream the reporter gene.
- Binding of the activator triggers transcription of the reporter gene (e.g. Luciferace) -> Quantification of the gene’s product (amount of light produced) to check whether your gene is transcribed
On this system you add your potential regulator.
- If it functions as an activator -> Light.
- Not activating -> No light.
The domain swap assay
Demonstrated the functional separation between DBD and AD. Based on reporter assay.
You have a reporter gene, lacZ, and a Gal4 site.
- Apply wild-type Gal4 -> Gal4 binds to the Gal4 site and activates the reporter LacZ -> LacZ shows that the transcription is on
- Truncated gal4 activator with removal of activation function -> Although Gal4 binds to the site, there is no transcription of LacZ. Demonstrating that you need the activation function for transcription, DNA-binding is not enough.
- We have another binding site, LexA site (binding site for activator LexA). Only DNA-binding domain of lexA -> No activation of reporter.
= Gal4 or LexA DBD can bind the DNA alone but do not trigger transcription of reporter gene LacZ
- We have another binding site, LexA site (binding site for activator LexA). Fusion of Gal4 activation domain with LexA DBD -> Activation.
Demonstrates that these two functions don’t have to come from the same protein. You can pick another activation domain, fuse it to the DNA-binding domain of another activation, and you will get transcription.
DNA-binding domains (DBDs)
Prokaryotic transcriptional regulators bind specific DNA sequences as homodimers via a helix-turn-helix domain (looks like an “L” lying across the DNA molecule)
- Helix 1 -> DNA major groove, sequence-specific interaction = Recognition helix
- Helix 2 -> DNA backbone
Same principal in eukaryotes, hovwever
- Eukaryotic regulators bind the DNA as homodimers, heterodimers (two different activators together at the same binding site) and rarely monomers
- Larger variety of DBD structures
- Extended range of DNA-binding sequence specificity
- This combination of possibilities will allow for more complexity and integration of more complex signals
What are some different types/structures of DNA-binding domains (DBDs)? Name 5
- Homeodomain proteins
- Zinc-containing DBDs
- Leucin zipper
- Basic Helix-loop-Helix (bHLH) proteins
- High-mobility group (HMG) proteins
Homeodomain proteins - structure, found where
Discovered in 1938 in Drosophila during study of homeotic transformation (replacement of body part with another)
- Regulate gene expression and cell differentiation during early embryogenesis -> Morphogenesis
- Alteration of homeodomain proteins cause developmental disorders (e.g. legs on head of fly)
When studying this, they discovered that the transcriptional regulators called homeodomain proteins, and that they contain some DNA-binding site very specific for these proteins.
- Consists of 3 alpha-helices
- H1 and H2 form a helix-turn-helix (HTH) domain
- H3 -> Perpendicular to HTH of H1 and H2 -> Interacts with major groove of DNA
Each of the homeodomain proteins in Drosophila will trigger the gene expression network responsible for development for each segment.
Zinc-containing DNA-binding domains - structure, found where
Discovered in 1985 in Xenopus during study of GTF TFIIIA’s DNA-binding capacities.
Various forms of Zinc-containing DBDs:
- Mainly Zinc fingers (Gal4, CTCF) - most common
- Zinc clusters
Structure and DNA-binding of zinc fingers:
- Classical Zinc finger = 4 amino acids, 2 Cys and 2 His (C2H2), stabilized by a Zinc ion in the middle (keeping the structure of the DBD together) -> Star-shaped
- 1 alpha-helix: Recognition helix -> Interaction with the major groove of DNA in a sequence-specific manner
- 1 antiparallell beta-sheet (2 beta-strands)
Many proteins (such as CTCF) feature a series of several Zinc fingers, one binding site after another. CTCF has 10 of these binding sites in series, which creates a very strong interaction with the DNA.
