200B, Jan 25-Feb1, 2005 Mechanisms of Transcriptional Activation
Lecture 1
Mechanisms of transcriptional activation
Intro-
In the next two lectures, and in the discussion next Monday, we’ll take a look at how our present understanding of the mechanisms by which transcriptional activators work was formed.
Original work performed in prokaryotes, where the predominant form of regulation appears to be negative (repression), rather than positive (activation). This is the opposite of the situation in eukaryotes.
However, the following paradigm emerged from this early work:
Steps in transcription:
1. promoter location
2. polymerase recruitment
3. promoter melting
4. initiation
5. escape
6. elongation
7. termination
In prokaryotic systems, there are examples where each of these steps is the target of regulatory events in various genes.
What is known about the regulators?
In contrast to what you have learned about eukaryotic regulators, the first prokaryotic reglators that were studied in detail were not necessarily modular in their organization. However, activation and DNA binding occurred on different surfaces of the molecule. Can change the DNA binding specificity without changing function of molecule and vice versa via swap experiments. (i.e. Ptashne's expts switching lambda and cro specificity)
How do the regulators work?
Two general classes of models:
1. By binding DNA, the regulator causes a change in the structure of the DNA that is transmitted to the promoter and affects the activity of RNA polymerase.
2. The regulator binds the DNA and then touches some other protein to change the rate of transcription.
a. the regulator could displace some other protein in the act of DNA binding.
b. the regulator could alter the activity of polymerase
-stabilize/destabilize an intermediate
-deliver a regulator e.g. a regulatory kinase
c. the regulator could recruit/repell components of the txn apparatus-in its simplest form, RNA polymerase itself
early expts gave good evidence for models 2a, 2c, later on 2b (stabilize intermediates/prevent initiation or escape).
So what about Euks?
To review, typical organization of eukaryotic promoter includes a core promoter with a TATA box and Inr, and an upstream region, the proximal promoter that serves as a target for multiple regulators.
Late 60’s/early 70’s eukaryotes have 3 distinct classes of nuclear rna polymerases.
In vitro transcription reactions developed in late 70’s.
Identification of basal or general transcription factors, Roeder lab played the key role in defining, early 80’s, Kornberg, Reinberg, Conaways, Tjian and others played roles in purification and cloning of subunits.
Go over strategies for purification----O/H
Show O/H illustrating a typical purification scheme.
need an assay for protein or activity
stress the importance of quantitation and discuss a purification table—O/H
Today, it is possible to assemble an in vitro reaction composed of purified RNA pol II and recombinant general factors.
So, using either a crude or purified transcription reaction can observe high levels of basal transcription (define this). point out that basal txn is a concept that is largely applicable to in vitro situation, chromatin likely represses all transcription in absence of activator in vivo. fuzzy terms in the literature
Now, how about activated transcription?????
First, what does a eukaryotic activator look like??
Brent and Ptashne Cell 1985
Had previously isolated pc (positive control) mutants in lambda repressor (which can normally activate as well as repress txn) that bind DNA but fail to activate txn. Thus in E. Coli regulators don’t appear to affect txn by altering DNA structure.
Now, can DNA binding and txn regulatory functions of the eukaryotic Gal4 protein be separated?
Show O/H
(controls show that these fusions can bind LexA sites in E. coli)
Conclusions
1. DNA binding and transcription activation are separable functions
2. nothing special about DNA binding domain, the lexA domain is not derived from euk.s but is perfectly capable of directing DNA binding in euks.
These expts imply that the function of DNA-binding domain is to tether the activation domain in the vicinity of promoter.
Further tests.....O/Hs
topologically link but physically separate activator from promoter
physically link bound activator via a protein bridge
Ma and Ptashne Cell 48
O/H extensive deletion mutagenesis of Gal4 identifies 2 activation domains (had previously mapped the DNA binding domain to 1st 147 amino acids)
O/H activation domains have no clear structure, no clear homology to one another or to only other mapped activation region at the time, but are rich in acidic amino acids
Ma and Ptashne Cell 51 O/H
try to identify essential features of activation domains by cloning random DNA fragments downstream of Gal4 DBD and identify those that can activate txn.
observe: about 1% of fragments activated, domains are small, have no obvious homology to one another other than acidic character
Later work showed that acidic residues might not even be that impt. So far, no activation domain has been xstalized, biophysical studies suggest they are flexible. Attempts to co-crystallize with target proteins have not been successful. Other types of activation domains appear to have equally nebulous characters-proline rich, glutamine rich. We clearly need some fresh ideas here.
How do eukaryotic activator proteins work?
Simplify the question to.....What do they touch?
The assumption, based upon previous work in prok.s, was that they were touching some part of the transcription apparatus, perhaps RNAP itself.
Gill and Ptashne
Overexpressed Gal4 in yeast. Found that reporter genes that lacked Gal4 binding sites were repressed.
O/H of table 2
Basic points Reporters have indicated UAS (enhancer) driving expression of a Gal1 core promoter fused to a Lac Z reporter.
-high Gal4 inhibits UASh driven reporter
-weakened derivatives of Gal4 result in weaker inhibition
-DBD is dispensible for inhibition (79-881 deriv)
-overexpression of the Gal 80 repressor of Gal 4 reverses the inhibition
conclude: activation domain of Gal4 deriv. is competing with factors bound to UASh for some limiting component of the txn apparatus
prediction: at very high levels of activator should self-squelch---they see this
Table shows effects of high copy (20-40) Gal4 expression vectors with strong ADH1 promoter
Not shown! low copy (1 or 2) Gal4 expression vectors with strong ADH1 promoter 43 U with UASh and ~3200 with UASG
this phenomenon, called squelching, has been observed in vitro as well.
This hypothetical limiting factor came to be called a co-activator.
What is this limiting target of activators?
In in vitro studies with highly purified factors see poor activation. In particular, if used recombinant TBP rather than paritally purified TFIID (which was very hard to purify to a satisfactory level) see that activation is lost. This led to the idea the TAFIIs are the missing coactivator.
Dynlacht, Hoey and Tjian
O/H Partially purify GTF’s from Drosophila and show that can reconstitute activated transcription in vitro. If use TBP rather than TFIID, no activation, but still have basal levels of transcription. Conclude non-TBP components of TFIID are the co-activators
O/H broke apart their partially purified TFIID and separated TBP from other proteins. now see only basal level transcription with TBP, no transcription with TBP-associated factors. If mix TBP and associated factors back together, recover activated transcription.
Preparative anti-TBP IPs pulls down TBP + 6 other proteins.
==TAFs!
conclude: TBP+TAFs gives response to activators, i.e. the TAFs are the missing coactivator.
Chen et. al.
Now, want to zero in on specific activator-coactivator contacts. They cloned and characterized 8 TAFIIs. Here they have expressed recombinant TAFIIs and have devised a way to reassemble TFIID and TFIID subcomplexes from recombinant proteins (i.e. they have devised an in vitro assembly pathway).
O/H of assembly scheme
Use these partially purified TFIIDs along with partially purified GTFs in in vitro txn expts +/- activators
Observe: O/H SP1 binds to and requires TAF110 for activation.
NTF-1 binds to TAF60 and TAF150, and either is
Sufficient for activation by NTF-1.
Conclude: Specific TAFIIs bind to and serve as targets for specific activators. TAFIIs=coactivators
Destroying the Paradigm
Fall of the TFIID as Target of Activators Model
1. Genetic studies had begun to challenge the TAF as coactivator model.
What genetic results would you predict?
in yeast, took Ts- (lethal) TAFII mutants, shifted to nonpermissive temp, prep’d RNA after 45 min.s and did whole genome microarrays--observed that only a minority of genes were TAFII dependent.
2. Michael Green’s lab had id’d yeast genes dependent upon the large TAF. They mapped the promoters of these genes to id the TAF dependent part and found that the core promoter (INR?) conferred TAF dependence. If the TAFs were the target of activators, would expect activator binding sites to confer dependence!
Not compatible with coactivator model
3. Oelgeschlager et al
They re-examine the model from a biochemical point of view.
Idea is to deplete crude extracts of TFIID with anti-TBP and anti-TAF affinity
chromatography.
Overhead--they were able to deplete some, but not all TAFIIs why?
-you will hear from JT that some TAFIIs appear in multiple complexes, whereas others are TFIID specific-they depleted IID specific TAFIIs
Now, perform in vitro transcription adding back TBP or partially prufied TFIID, see that either one will support activated transcription with the strong Gal4-VP16 activator.
See that a proline rich activator is also TAFII independent
How could variations in experimental protocol explain these differences?
Will come back to final expt. later
======
Starting Over, What is the Limiting Factor the Interacts with Activators?
Strategy: go back to squelching assay, can it be used as an assay to purify the factor?
remember that Tij’s TFIID argument was based upon using partially purified fractions for in vitro transcription experiments, not upon setting up an unbiased assay for activation and using this to demonstrate a requirement for or as the basis of an assay to purify the coactivator
Kelleher et al
1. see squelching in vivo at multiple genes Overhead
2. See squelching in vitro Overhead
-note auto-squelching at GAL10 UAS
-squelching was AD dependent
3. Used this assay to identify fractions from DEAE column that overcome squelching O/H
-Fig. shows trancription levels with squelched reactions (eluate overcomes squelching)
note importance of having a quantitative assay for following activity, this will be critical later
Potential explanations? Ask them! Overhead
a. adding extra activator (T-richBP)
-no, don’t see squelching of a Gal4 template
would expect if adding xs of another act.
b. non-specific binding of Gal4-VP16
-no, activation by Gal4-VP16 not disrupted
c. general stimulatory effect
-no, activation depends upon Gal4-VP16
This factor is not a previously id’d basal factor
-adding them back doesn’t overcome squelching (data not shown)
Flanagan et al
Now rather using crude extracts and squelching as an assay, use purified transcription factors to perform in vitro transcription does mediator reconstitute response to activators?
OVERHEAD
1. Purified factors alone are not responsive to activator
2. The same column fraction that overcomes squelching in vitro, restores activator response in vitro
3. GTF’s and Pol II don’t substitute for this factor, TBP alone rather than TFIID is sufficient for this function.
Is this a Gal4 specific phenomenon?
No, the same fraction also supports activation by GCN4
OVERHEAD
Gal4 -VP16 squelches Gcn4 and vice versa
Suggests that both activators use the same mediator.
Named this fraction “Mediator”
Now, purify mediator fraction
Kim et al
OVERHEAD
mediator copurifies with RNA Pol II
-saw that in a purification, Pol II factionates in two peaks, one of which contains mediator activity.
OVERHEAD
could separate Pol II from mediator on an anti-CTD column
-mediator+Pol II (+TFIIF) = “holopolymerase”
activities of mediator--on board
1. stimulates basal transcription
2. stimulated CTD kinase activity of TFIIH
3. required for activated transcription
all basal factors, including TFIID, are still required for transcription
Purified to homogeneity
holopol II- > or = 32 proteins
-12 pol II subunits
-3 TFIIF subunits
-17 others (more found later)
--Srbs
- Gal 11, Sug1 previously implicated in txn
interaction with pol II appears to be through the CTD
-anti CTD antibody elutes mediator from core Pol II
Srb complex: SRB genes previously implicated in CTD and activator function by Young lab
- identified as genetic suppressors of CTD truncation mutations
-could purify a complex containing Srb proteins that is similar in composition and activity to Kornberg’s holopol II complex (young’s also has IIB and IIH)
-Srb proteins likely essential for transcription of most or all genes in vivo (in contrast to TAFIIs!)
Myers et al (G&D, 1998)—O/H
-identify additional mediator proteins
-mediator directly binds to a CTD peptide
-Pol IIb does not respond to mediator therefore mediator fn requires the CTD
Now, if mediator is target of activators, what can we predict about activator-mediator interactions?
Hengartner et al
- does holopol II bind/interact with activators?
OVERHEAD
Test by mixing VP16 or non-activating VP16 mutant with holopol II and IP’ing with anti-Srb5 antibody
WT, but not mutant VP-16 co-IPs
OVERHEAD
2nd test of same point
make GST-VP16 columns and put holo-pol II over
see interactions once again, but TBP and IIB also there
Since VP16 previously shown to touch both of these, the interaction with holo-polII could be through these two basal factors.
Therefore, is pol II or TBP or other basal factor required?
prepare Mediator according to Kornberg lab protocol and test
OVERHEAD
In this figure, we see that pol II and TBP are not required for this interaction
there is evidence for similar complex(es) in humans
Fig from Oeglsechlager
anti-hSrb7 depletion results in loss of activation in vitro relate back to TFIID depletions
where is the add back control?
since this paper, many labs have purified complexes from humans that resemble mediator
show O/H from review………
The new standard model
OVERHEAD from young paper
Do we believe this?
We’ve been burned by this sort of combination of binding and functional assays before-is anything new here?
-activation deficient mutants don’t bind--Hengartner
What about genetics?
-mutations in some mediators subunits known to cause activation defects
-e.g. gal11 mutants are defective for Gal txn
What would you consider to be good evidence?
-srb4- no pol II txn. good enough?