A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor

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A eukaryotic transcriptional activator bearing
the DNA specificity of a prokaryotic repressor

• Roger Brent and Mark Ptashne

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What is the GAL pathway?
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How does Gal4p work?
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What is the general question?

• How do transcription factors function?
• Do all transcription factors function in the same
  way?
• Are there some basic functions of transcription
  factors and what are they?

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What is the general approach

• Molecular biology
• Why use molecular techniques here?
• Difficult to use classical genetics to analyze a
  single protein this is better for pathways or
  interactions, etc.
• Easy to ask cross-species, cross-phylogenetic
  domain questions
• Allows specific changes to be tested

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UAS

• Upstream activation sequences
• Sequences of DNA that bind RNA polymerase II
  transcription factors
• Often named for the factor that binds them, e.g.
  GAL4 UAS
• These are involved in up-regulation of gene
  expression
• They have a consensus sequence
• Similar to enhancers in higher eukaryotes

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GAL4 UAS

• Gal4p is bound when cells are grown on galactose
• In the absence of galactose, Gal4p is bound and
  inhibited by Gal80p
• Consensus sequence

CGGASSACWGTSSTCCGWRS CGGattAgAagcCgCCGAG
CGcgccgCACTgCTCCGAA CGGgtgACAGccCTCCGAA
aGGaagACTCTcCTCCGTG CGGggcggAtcaCTCCGAA
CGGcggtCTtTcGTCCGTG CGcgccgCACTgCTCCGAA
Actual sequences
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Whats the question?

• Two hypotheses for how transcription factors
  worked
• 1) Gal4p binds to DNA in a way that stabilizes a
  unique structure or confimration, e.g.
  left-handed DNA. The perturbed structure then
  would be transmitted down the helix and help
  proteins bind near the transcription start
• 2) Gal4p contacts the DNA without perturbing the
  structure and activation of transcription happens
  when Gal4p binds other proteins (or recruits
  them).
• Brent wanted to know if he could separate the DNA
  binding from the transcription activation of
  Gal4p.

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What was known?

• In E. coli the lambda repressor was known to bind
  to the repressor site and recruit RNA polymerase
  by touching it.
• lexA was known to bind as a dimer and had two
  domains one that bound DNA and the other that
  is involved in protein-protein interactions
  (dimerization) that were joined by a hinge
  region.
• Guarante had isoltated a lamda repressor that
  bound DNA but couldnt activate transcription.
• Roger had just synthesized lexA in yeast and
  shown that it could bind to the operator and, if
  placed between the UAS and the transcription
  start site, could repress transcription.

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What is the specific approach
1) Make fusion gene that will encode an in-frame
lexA DNA-binding domain fusion with various parts
of Gal4p making a fusion or chimeric protein
This promoter works in E. coli.It has a bacterial
orgin and selectable marker. Where are they?
This promoter works in yeast. It has a yeast
selectable marker (LEU2) and a yeast origin of
replication (2 micron ori ?high copy number
plasmid.
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Eucaryotic gene structure

• Get picture

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Details of fusion construct

• Made lexA-GAL4, it could be transcribed and
  synthesized in both E. coli and yeast.
• Amino terminal 87 aa from lexA
• Carboxy terminal 807 aa from Gal4p (of 884 aa)
• NO PCR so had to get fragments the hard way,
  and ligate them into a plasmid.

Get diagram of Gal4p domains
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Does the fusion work in E. coli?
b-galactosidase is a reporter gene used to
quantify transcription. It is encoded by the
lacZ gene. IPTG inactivates lac repressor LexA
is its own repressor
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Constructs to test if the fusion works in yeast

1. Normal UAS, GAL1p, lacZ reporter
2. GAL1 w/o UAS
3. lexA operator
4. 17-mer of lexA operator
5. lexA operator 3 of GAL4 UAS
6. No UAS, CYC1 promoter
7. lexA operator, CYC1p
8. lexA operator, more 5, CYC1p
9. UASG 5 of CYC1 promoter
10. 17mer, CYC1 promoter

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The lexA-GAL4 fusion is able to activate
expression from a GAL1 promoter containing only
the lexA operator.
The lexA-GAL4 fusion works With a lexA
operator But also with cyc1 UAS and with a GAL4
17-mer. Question is the Gal4 part enough to
activate from all or part of the GAL4 UAS?
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Were the start sites the same?
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Does the fusion protein activate from a GAL4 UAS?
GAL1 was activated from the GAL4 UAS in the
presence of the fusion protein. What
happened? Suggests that there is enough
carboxy-terminus to titrate Gal80. Why cant it
just be active?
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Discussion/conlusions
Can separate DNA-binding and transcription
activation. Transcription factor funtion is
conserved between procaryotes and
eucaryotes Since it doesnt matter which factor
binds the DNA, there must not be a
factor-specific change in DNA confirmation.
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Discussion/conclusions

• LexA-Gal4p fusion activates transcription but the
  LexA alone does not. This is consistent with the
  hypothesis that specificity for gene activation
  is not in the DNA-binding domain.
• Found that first 74 aa of Gal4p bind DNA but
  dont activate transcription.

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Other observations

• Binding of Gal4p to a 17-mer that has
  approximately two-fold rotational symmetry (10
  bases per turn), suggests it binds as a dimer.
• Made a lexA-GCN4 hybrid that also works.
• Says this could be a tool for studying
  transcription