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

1. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor Roger Brent and Mark Ptashne

2. What is the GAL pathway?

3. How does Gal4p work?

4. 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?

5. 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

6. 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

7. 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: CGGASSACWGTSSTCCGWRSCGGattAgAagcCgCCGAGCGcgccgCACTgCTCCGAACGGgtgACAGccCTCCGAAaGGaagACTCTcCTCCGTGCGGggcggAtcaCTCCGAACGGcggtCTtTcGTCCGTGCGcgccgCACTgCTCCGAA Actual sequences

8. What’s 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.

9. 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 couldn’t 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.

10. 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.

11. Eucaryotic gene structure • Get picture

12. 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

13. 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

14. Constructs to test if the fusion works in yeast • Normal UAS, GAL1p, lacZ reporter • GAL1 w/o UAS • lexA operator • 17-mer of lexA operator • lexA operator 3’ of GAL4 UAS • No UAS, CYC1 promoter • lexA operator, CYC1p • lexA operator, more 5’, CYC1p • UASG 5’ of CYC1 promoter • 17mer, CYC1 promoter

15. 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?

16. Were the start sites the same?

17. 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 can’t it just be active?

18. Discussion/conlusions Can separate DNA-binding and transcription activation. Transcription factor funtion is conserved between procaryotes and eucaryotes Since it doesn’t matter which factor binds the DNA, there must not be a factor-specific change in DNA confirmation.

19. 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 don’t activate transcription.

20. 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