BIO120H1 Lecture Notes - Lecture 11: Plasmid, Dna-Binding Domain, Tryptophan

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2 Feb 2013
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Lecture 11: Protein-protein interactions
- Let’s take stock: we’ve talked about the transcriptome, its involvement
b/w organisms even within an organism. We’ve talked about
transcription factor use, which gene regulatory proteins are used at
which points in time & we’re going to determine ultimately what the
transcriptome is that gets made in those cells. We took a look at the fact
that the proteins that ultimately get made from the transcriptome is
going to give rise to those differences those proteins don’t function in
isolation (E2-E3 ubiquitin ligase complex 2 proteins working together
to perform an important cellular function). Proteins are functioning
together in complexes that really drives differences b/w cells which is
what we’re going to focus on in today’s lecture.
Interactome – interactions b/w proteins
Complete set of protein-protein interactions produced by the proteome
at any one time.
Yeast 2-hybrid assay
- Helps us define, not just a few interactions b/w proteins but a good
many interactions b/w proteins. In fact, all that occur within cells.
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- Stan Fields developed the yeast 2-hybrid assay.
- The yeast two-hybrid assay takes advantage of the way in which
eucaryotic transcription factors/gene regulatory proteins work.
Identify binding partners – 2 proteins that interact with each other.
- Looking for the interaction b/w the BAIT protein & its corresponding
PREY, can be thought of protein 1 and protein 2 & testing the
hypothesis do those proteins interact with each other?
- System takes advantage of a transcription factor that has been split in
half, DNA binding domain on BAIT & transcriptional activation domain
on PREY separating those 2 activities so the protein no longer functions
as one protein independently but requires those 2 domains to be brought
together by the interaction b/w the 2 proteins to eventually activate
transcription. How does that all work?
- Starts with transcription factor called GAL4.
- GAL4 has a DNA-binding domain DBD and a transcription-activation
domain.
- UASG – upstream activator sequence.
- So when galactose is present, GAL4 binds to this upstream activator
sequence that is found upstream of a minimal promoter & it activates a
gene encoding galactose utilizing enzyme so there are coactivators and
RNA polymerase II, have the mRNA made and the corresponding
enzyme.
- Like other eucaryotic transcription factors, it turns out that the DBD &
the TAD are separable & can function on their own, that is, the DNA
binding domain can bind DNA in the absence of the activation domain.
The activation domain provided that it is tethered to a DNA binding
domain will function as an activation domain. It turns out that the
activation domain needs to be somehow be associated with the DNA
binding domain to do its job.
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Bait
Prey
- They identified by doing a number of deletions what portion of the
sequence was the DNA binding domain so they know that DBD must
reside upstream of this particular sequence b/c if deleted, it loses DNA
binding activity. By deleting from the carboxy terminus, they identified
similarly the region that’s necessary for transcription (TAD).
- They’ve done internal deletions, & shown that by reducing proteins to
this little stretch of 74 AAs here & a little under 120 AAs, that you have
both DNA binding activity & transcriptional activation showing that
those 2 bits are the important bits.
- For the yeast 2-hybrid assay, what’s happened is that these 2 pieces
have been separated so that you still have DNA binding that can occur
b/c you have the DNA binding domain but b/c the transcriptional
activation domain has been separated from it, there is no longer
transcriptional activation. What’s been done is to fuse a BAIT protein,
one protein of interest to the DNA-binding domain & a so called PREY
protein in translational fusion with the transcriptional activation domain
if the 2 proteins interact, they will bring the DBD in close enough
proximity to the TAD so now we acquire DNA binding activity &
transcriptional activity & we’re able to activate a corresponding target
gene.
- If BAIT and PREY interact they bring the DBD & the TAD into close
enough proximity that they’re able to activate transcriptional activity
from genes containing the UASG.
- All these components are encoded on plasmids.
- In the 1st instance, we’ve got a promoter driving the expression of
DNA that encodes the GAL4 DBD & this is upstream of our gene of
interest that encodes our protein of interest, the BAIT. Together they’re
going to make a fusion protein which is the BAIT comprising the DBD
plus the protein of interest.
- In yeast, in order to make sure that our plasmid is there, we need to
include an extra gene so it’s a promoter driving the expression of the
TRP gene.
Selectable marker
- Yeast cells with the plasmid b/c they have the TRP gene on the
plasmid will enable the cell to make tryptophan & you don’t need to
have tryptophan in the medium.
- This way we make sure that only cells that have the plasmid are the
living cells – the living cells are the only cells that can possibly have the
plasmid – make sure that our plasmid is there making our fusion protein.
- TRP gene is a selectable marker that ensures that we’ve got a plasmid
present that is going to make our BAIT.
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