CAP binds DNA in the presence of cAMP from (Heyduk_Biochem_1989) No cAMP 100uM cAMP 10mM cAMP No...

CAP binds DNA in the presence of cAMP

from (Heyduk_Biochem_1989)

No cAMP

100uM cAMP

10mM cAMP

No binding withno cAMP, bestbinding with 100 uMcAMP.

Increasing CAP from lane A to lane D

Muller-Hill states and others thought thatCAP 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.

McKay and Stietz think that CAP binds left handed DNA

CAP binding to DNA

CAP bends right-handed DNA by ~ 90o

Heliix F

cAMP levels are the same in glucose and lactose

cAMP spikes as glucose runs out

Addition of cAMP alleviates diauxie, but -gal is still repressed when

glucose is present

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!)

lacZ is kept off because glucose inhibits transport of lactose

The PTS (phophotransferase system) in bacteria

PTS function in E. coli

turns on lacand othergenes

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?

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

How does cAMP/CAP effect transcription?

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

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

Extragenic suppression: physically interacting proteins

Example: Complexes that bind nutrients in from the outside

CAP w/o cAMP

Red: DNA binding helix

Yellow: hinge region whereDNA binding domain swingsinto place

Blue: residues that canmutate to give a CAP thatno longer needs cAMP tobe active.

CAP w/ cAMP bound to DNA

Red: DNA binding helix

Yellow: hinge region whereDNA binding domain swingsinto place

Blue: residues that canmutate to give a CAP thatno longer needs cAMP tobe active.

Domain 1

Schematic of sigma70

Murakami et al. 2003 Curr. Op. Struct. Biol. 13:31

Domain 2Binds -10

Domain 3 Domain 4Binds -35

D2.4 contacts the -10 site and confers specificity

-35 region

-10 region

red-sigma

green= Beta, Beta’

tan=alpha I, alpha II

Thermus aquaticusRNA polymerase

Murakami et al. (2002) Science 296:

Building a consensus for the 32 binding site

DNA-with no RNAP binding site (promoter)

DNA +increasing RNAPNo CAP

DNA +increasing RNAP+ CAP(w.t)

Same as “a” but CAP is mutant(binds DNA, but doesn’t activateSquares: No CAPCircles: With mutant CAP

Fluorescence polarization increaseswhen proteins slow down DNA movement in solution

From Heyduck_Nature_1993

Modes of transcriptional activation

Activator interacts withsubunits and helpsRNAP bind

Activator interacts withsubunit domain 4 and helps RNAP bind

Activator interacts between -35 and -10 andreorients these sites forbetter RNAP binding

Genes activated by CAP in MG1655

Red= genes transcribed more when CAP•cAMP is presntYellow= expression same withand without CAP•cAMPGreen=Less expression withCAP•cAMP

center line: equal expression withand without CAP•cAMP

outer lines: mark 2x differencebetween w/ an w/o CAP•cAMPsee arrow on diagram

100 units w/o CAP•cAMP

200

units

w/ C

AP•c

AMP

Genes not activated by the HL159 mutant are missing—these are regulated by type

I activationLots of missing red dots. These aregenes that need type I activationwhich CRP HL159 can’t do.

Genes not activated by the KE101 mutant are missing—these are regulated by type

II activationSome missing red dots. These aregenes that need type II activationwhich CRP KE101 can’t do.

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

So, what does cAMP do?

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

Extragenic suppression: biochemically interacting proteins

-35 region

-10 region

red-sigma

green= Beta, Beta’

tan=alpha I, alpha II

Thermus aquaticusRNA polymerase

Murakami et al. (2002) Science 296:

Modes of transcriptional repression

Repressor directly blocksRNAP binding

Repressors form a loopIn DNA and blockRNAP binding

Modes of transcriptional repression

Repressor alters thefunction of an activator

CAP w/ cAMP bound to DNA

Red: DNA binding helix

Yellow: hinge region whereDNA binding domain swingsinto place

Blue: residues that canmutate to give a CAP thatno longer needs cAMP tobe active.

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