cap binds dna in the presence of camp from (heyduk_biochem_1989) no camp 100um camp 10mm camp no...
DESCRIPTION
McKay and Stietz think that CAP binds left handed DNATRANSCRIPT
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