Turning  Cro into a Transcriptional Activator

1. Turning  Cro into a Transcriptional Activator Fred Bushman and Mark Ptashne Cell (1988) 54:191-197 Presented by Natalie Kuldell for 20.902 February 4th, 2009

2. Small patch of acidic residues is necessary and sufficient for transcriptional activation Figure 1 cI normally activates transcription cro normally represses transcription cro/cI chimera activates transcription!

3. Site-directed mutagenesis of cro helix to make acidic patch cartoon ofcI binding DNA Figure 2 Thr17 Lys21 Asp22 Tyr26 fig from “A Genetic Switch” 4 amino acid substitution --> “cro67”

4. Why might this work?

5. Site-directed mutagenesis of cro helix to make acidic patch Figure 2 Thr17 Lys21 Asp22 Tyr26 4 amino acid substitution --> “cro67”

6. Site-directed mutagenesis of cro helix to make acidic patch Figure 2 Thr17 Lys21 Asp22 Tyr26 4 amino acid substitution --> “cro67”

7. Site-directed mutagenesis of cro helix to make acidic patch Figure 2 Thr17 Lys21 Asp22 Tyr26 4 amino acid substitution --> “cro67”

8. Site-directed mutagenesis of cro helix to make acidic patch Figure 2 Thr17 Lys21 Asp22 Tyr26 4 amino acid substitution --> “cro67”

9. Protein -helix recognizes sequence in DNA major groove model of lac repressor binding lac operator http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=35&theme=printer

10. Protein -helix recognizes sequence in DNA major groove • Wild type cro • binds OR3>>OR2 = OR1 • binding to OR3 shuts off tx’n from PRM • Wild type cI • binds OR1>OR2>OR3 • binding to OR2 activates tx’n from PRM

11. OR1>OR2>OR3 Figure 3 Protein -helix recognizes sequence in DNA major groove • Wild type cro • binds OR3>>OR2 = OR1 • binding to OR3 shuts off tx’n from PRM • Wild type cI • binds OR1>OR2>OR3 • binding to OR2 activates tx’n from PRM • cro67 • binds? • activates?

12. OR1=OR2>OR3 Figure 3 Protein -helix recognizes sequence in DNA major groove • Wild type cro • binds OR3>>OR2 = OR1 • binding to OR3 shuts off tx’n from PRM • Wild type cI • binds OR1>OR2>OR3 • binding to OR2 activates tx’n from PRM • cro67 • binds? • activates?

13. 395 bases 250 bases cro67 activates transcription in vitro Figure 4 [cro67] 0 In vitro tx’n rxn’s + buffer + DNA w/ PRM + PR cro67 (purified) + 32P-ATP, CTP, GTP or UTP 37° 10’ then + RNAP 37° 10’ then +formamide to gel

14. ~5x 395 bases ~5x 250 bases cro67 activates transcription in vitro Figure 4 cut out bands and count Observe: txn of PR as txn of PRM when cro67 added Q’s: What are extra bands? Is cro67 bound in natural way?

15. OR1=OR2>OR3 cro67 binds operator sequences as expected Figure 4 [cro67] 0 DNase footprint + buffer + 32P-DNA w/ PRM + PR cro67 (purified) 37° 10’? then + DNase 37° 10’? then +formamide to gel? Observe: Q: is assay sensitive to different conformations of bound prot?

16. cro67 activates transcription in vitro Supporting data/controls Figure 5 Wild type cro does not activate txn in vitro using in vitro txn rxn, DNase ftpt Figure 6 cro67 does not activate txn from other promoters cro67 in vivo exp’ts hampered by low affinity for operators (~100x < wt cro)

17. Summary of 434 cI data ** in vivo (-gal assays on lysogen) **in vivo DMS ftpt ** in vitro txn rxns, DNase ftpt

18. Turning cro into a transcriptional activator key assumption in vitro conclusions have meaning in vivo biggest mistake mixing the 434 work in not pushing in vivo work significance/meta-lessons • protein engineering by analogy (cro is like cI, thus…) • small changes (e.g., individual AAs) are important • good data enables thoughtful experiments • be open to surprises (e.g., DNA binding) • ask the next question: does activation work the same way in eukaryotic cells?