1. CAP binds DNA in the presence of cAMP from (Heyduk_Biochem_1989) No cAMP 100uM cAMP 10mM cAMP No binding with no cAMP, best binding with 100 uM cAMP. Increasing CAP from lane A to lane D

2. Muller-Hill states and others thought that CAP dimer with one bound cAMP bound DNA best (seen at 100 uM) and that at 10 mM cAMP two monomers bound cAMP and that decreased affinity for DNA. Steitz showed that it was likely that CAP bound 2 cAMPs at 100 uM and 4 cAMPs (perhaps abnormally) at 1 mM.

3. McKay and Stietz think that CAP binds left handed DNA

4. CAP binding to DNA

5. CAP bends right-handed DNA by ~ 90 o Heliix F

6. cAMP levels are the same in glucose and lactose

7. cAMP spikes as glucose runs out

8. Addition of cAMP alleviates diauxie, but  - gal is still repressed when glucose is present

9. If  -gal is not made when glucose is present, even when cAMP is present, then what is keeping it off (it’s not low cAMP!)

10. lacZ is kept off because glucose inhibits transport of lactose

11. The PTS (phophotransferase system) in bacteria

12. PTS function in E. coli turns on lac and other genes

13. So, if glucose transport stops lactose from getting into the cell, what is cAMP for? Why is it connected to glucose transport and what does it have to do with diauxie?

14. The depletion of glucose significantly increases intracellular concentration of the CRP-cAMP complex The increase in CRP-cAMP level should allow quick and efficient induction of lacZ and more importantly lacY. So, cAMP helps LacY be made quickly during the lag, allowing a shortened lag time, this allows quick induction of lacZ

15. How does cAMP/CAP effect transcription?

17. If you don’t know what the -35, -10 and +1 sites are, you need to read about them in Schleif text (pp 96-97, then page 95 on sigma factors)

18. Intragenic suppression An example: one amino acid change compensates for another Salt bridge hold domains together through +/- interactions Salt bridge destroyed by Asp to Lys mutation Second mutation allows salt bridge with mutant Lys

19. Extragenic suppression: physically interacting proteins Example: Complexes that bind nutrients in from the outside

20. CAP w/o cAMP Red: DNA binding helix Yellow: hinge region where DNA binding domain swings into place Blue: residues that can mutate to give a CAP that no longer needs cAMP to be active.

21. CAP w/ cAMP bound to DNA Red: DNA binding helix Yellow: hinge region where DNA binding domain swings into place Blue: residues that can mutate to give a CAP that no longer needs cAMP to be active.

22. Domain 1 Schematic of sigma70 Murakami et al. 2003 Curr. Op. Struct. Biol. 13:31 Domain 2 Binds -10 Domain 3Domain 4 Binds -35

23. D2.4 contacts the -10 site and confers specificity

24. -35 region -10 region red-sigma green= Beta, Beta’ tan=alpha I, alpha II Thermus aquaticus RNA polymerase Murakami et al. (2002) Science 296:

25. Building a consensus for the  32 binding site

26. DNA-with no RNAP binding site (promoter) DNA +increasing RNAP No CAP DNA +increasing RNAP + CAP(w.t) Same as “a” but CAP is mutant (binds DNA, but doesn’t activate Squares: No CAP Circles: With mutant CAP Fluorescence polarization increases when proteins slow down DNA movement in solution From Heyduck_Nature_1993

27. Modes of transcriptional activation Activator interacts with  subunits and helps RNAP bind Activator interacts with  subunit domain 4 and helps RNAP bind Activator interacts between -35 and -10 and reorients these sites for better RNAP binding

28. Genes activated by CAP in MG1655 Red= genes transcribed more when CAPcAMP is presnt Yellow= expression same with and without CAPcAMP Green=Less expression with CAPcAMP center line: equal expression with and without CAPcAMP outer lines: mark 2x difference between w/ an w/o CAPcAMP see arrow on diagram 100 units w/o CAPcAMP 200 units w/ CAPcAMP

29. Genes not activated by the HL159 mutant are missing—these are regulated by type I activation Lots of missing red dots. These are genes that need type I activation which CRP HL159 can’t do.

30. Genes not activated by the KE101 mutant are missing—these are regulated by type II activation Some missing red dots. These are genes that need type II activation which CRP KE101 can’t do.

31. Zheng’s ROMA data Down arrows in the HL/wt column indicate poor expression by the HL159 CRP (no type I) Down arrows in the KE/wt column indicate poor expression by the KE101 CRP (no type II) Horizontal bars mean no effect by HL159 or KE101 CRP

32. So, what does cAMP do?

33. CAP was thought to bind left-handed DNA CAP Cro To imagine CAP binding, just flop the protein, so that the F-helix is pressed against the DNA

34. Extragenic suppression: biochemically interacting proteins

35. -35 region -10 region red-sigma green= Beta, Beta’ tan=alpha I, alpha II Thermus aquaticus RNA polymerase Murakami et al. (2002) Science 296:

36. Modes of transcriptional repression Repressor directly blocks RNAP binding Repressors form a loop In DNA and block RNAP binding

37. Modes of transcriptional repression Repressor alters the function of an activator

38. CAP w/ cAMP bound to DNA Red: DNA binding helix Yellow: hinge region where DNA binding domain swings into place Blue: residues that can mutate to give a CAP that no longer needs cAMP to be active.

39. A molscript (4) ribbon drawing of the CAP dimer bound to DNA and the two cAMP molecules (magenta) per monomer, one labeled SYN and the other, ANTI. Passner J M, Steitz T A PNAS 1997;94:2843-2847 ©1997 by The National Academy of Sciences of the USA