ribbon drawing of the CAP dimer bound to DNA and the two cAMP

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CAP binds DNA in the presence of cAMP
from (Heyduk_Biochem_1989)
Increasing CAP from lane A to lane D
No cAMP
100uM cAMP
10mM cAMP
No binding with
no cAMP, best
binding with 100
uM
cAMP.
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.
McKay and Stietz think that CAP binds
left handed DNA
CAP
dimer
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 bgal is still repressed when glucose is present
If b-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 lac
and other
genes
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 in
Schleif 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 where
DNA binding domain swing
into place
Blue: residues that can
mutate to give a CAP that
no longer needs cAMP to
be active.
CAP w/ cAMP bound to DNA
Red: DNA binding helix
Yellow: hinge region where
DNA binding domain swing
into place
Blue: residues that can
mutate to give a CAP that
no longer needs cAMP to
be active.
Schematic of sigma70
Domain 1
Domain 2
Binds -10
Murakami et al. 2003 Curr. Op. Struct. Biol. 13:31
Domain 3
Domain 4
Binds -35
D2.4 contacts the -10 site and confers specificity
-35 region
Thermus aquaticus
RNA polymerase
tan=alpha I, alpha II
red-sigma
-10 region
green= Beta, Beta’
Murakami et al. (2002) Science 296:
Building a consensus for the 32 binding site
DNA-with no RNAP binding site
(promoter)
Fluorescence polarization increases
when proteins slow down DNA
movement in solution
DNA +increasing RNAP
+ CAP(w.t)
DNA +increasing RNAP
No CAP
From Heyduck_Nature_1993
Same as “a” but CAP is mutant
(binds DNA, but doesn’t activate
Squares: No CAP
Circles: With mutant CAP
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 an
reorients these sites for
better RNAP binding
200 units w/ CAP•cAMP
Genes activated by CAP in MG1655
Red= genes transcribed more
when CAP•cAMP is presnt
Yellow= expression same with
and without CAP•cAMP
Green=Less expression with
CAP•cAMP
center line: equal expression wit
and without CAP•cAMP
100 units w/o CAP•cAMP
outer lines: mark 2x difference
between w/ an w/o CAP•cAMP
see arrow on diagram
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.
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.
Zheng’s ROMA data
Down arrows in the HL/wt column indicate poor expression by the HL159 CRP (no type
Down arrows in the KE/wt column indicate poor expression by the KE101 CRP (no type
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
Thermus aquaticus
RNA polymerase
tan=alpha I, alpha II
red-sigma
-10 region
green= Beta, Beta’
Murakami et al. (2002) Science 296:
Modes of transcriptional repression
Repressor directly bloc
RNAP binding
Repressors form a loop
In DNA and block
RNAP binding
Modes of transcriptional repression
Repressor alters the
function of an activator
CAP w/ cAMP bound to DNA
Red: DNA binding helix
Yellow: hinge region where
DNA binding domain swing
into place
Blue: residues that can
mutate to give a CAP that
no longer needs cAMP to
be 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
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