Building the preinitiation complex

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Catabolite Activator Protein CAP mediates
catabolite repression in bacteria
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Activators CAP
80
CAP bends DNA ca. 80 deg.
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Activators CAP
CAP bends DNA approximately 80 deg
What are possible ramifications? -brings
together distant proteins -may help melt some
promoters
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Activators CAP
Rotation of the monomer subunits induced by cyAMP
binding.
Cyclic AMP binds to the N-terminal domain and
causes the two C helices to reorient and move
the DBDs apart.
C
C
Passner et al. (2000) J. Mol. Biol. 304 847-859.
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Activators CAP
The CAP activator can bind to different sites
with respect to the promoter.
1- distances must be integral number of DNA
turns. Suggests that CAP and RNA pol must be on
the same helical face of the DNA. 2- No
CAP-dependent promoter has a good -35 site. 3-
gal promoter CAP increases the transition from
the closed to open promoter complex. 4- lac
promoter CAP increases formation of the closed
promoter complex.
AraC
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CAP Fun Facts
1- Activates transcription at more than 100
promoters.
2- molecular mass of 45 kDa and binds DNA as a
dimer.
3- the first transcriptional activator to be
purified (1970) and the first to have 3D
structure determined.
4- At the simplest CAP-dependent promoters only 3
components are required CAP, RNA pol, and DNA.
Most bacterial and eukaryotic promoters are more
complicated.
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class I- require only CAP for activation and have
single site for CAP binding located upstream from
promoter. The CAP site can be at various
distances, but must be on the same helical face
as RNA pol. Example lac
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Activators CAP
CAP makes contact with the a-CTD
Class I CAP-dependent promoter example lac
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class I-
Activation region-1 (AR1) makes contact with the
a-CTD at the lac promoter.
Only contact with a single a-CTD is required.
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CAP Fun Facts
Note the a-CTD binds to the DNA immediately
downstream of CAP in both cases.
Class I-
DNA bending is not thought to play a role.
Mechanism interactions between CAP and a-CTD
increase binding constant for formation of the
closed promoter complex. Increases initiation by
facilitating recruitment of holoenzyme to the
promoter.
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class II- only require CAP for activation and
have a single CAP site that overlaps the promoter
replacing the -35 site. Example gal P1 promoter
-41.5
Note that the a-CTD binds upstream of the CAP in
Class II.
Busby Ebright (1999) J. Mol. Biol. 293 199-213.
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Activators CAP
gal operon
Class II
-CAP cAMP
Rifampicin NTPs
CAP cAMP
CAP promotes open promoter complex at the gal
promoter
Very poor -35 consensus in all CAP-dependent
promoters
Note open complex is not formed on this promoter
in the absence of NTs unless CAP is bound to the
promoter.
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CAP Fun Facts
Class II
Activation regions 1 and 2 (AR1, 2) make contact
with the a-CTD and the a-NTD, respectively, at
the gal promoter. AR2 is in the N-terminus near
the cyAMP binding domain. AR2 carries a net
positive charge of 2.
Busby Ebright (1999) J. Mol. Biol. 293 199-213.
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CAP Fun Facts
Class II
CAP makes contact with a single a-subunit. The a
that contacts b works better than the one that
contacts b.
NOTE the -35 site is absent, but region 4 of
sigma 70 binds here.
4
Busby Ebright (1999) J. Mol. Biol. 293 199-213.
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CAP Fun Facts
Class II
The upstream CAP makes contact with the a-CTD
with AR1.
The downstream CAP makes contact with the
N-terminal domain of a through AR2.
The a-CTD makes contact with the DNA upstream of
CAP. CAP is located in its preferred location.
Busby Ebright (1999) J. Mol. Biol. 293 199-213.
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class III- These require two or more CAP dimers,
or one or more CAP molecules with one or more
regulon-specific activators. Examples araBAD and
malK.
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class III-
Uses a class I mechanism of activation AR1
contact with the a-CTD.
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CAP Fun Facts
Three classes of CAP-dependent promoters
Class III-
Uses both class I and class II mechanisms of
activation AR1 contact with the a-CTD, AR2
contact with a-NTD.
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CAP Fun Facts
Class III- Sometimes coupled with a negative
regulatory mechanism called anti-activation.
CytR purine metabolism
A negative regulator may block the action of CAP
as seen in the case of the CytR promoter. CAP is
prevented from contacting the a-CTD and the CTD
is blocked from binding the DNA.
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CAP Fun Facts
Anti-activation can also occur with class I and
II promoters
Some bacteriophage use anti-activation to block
activity of E. coli RNA pol. T4 blocks CAP
interaction with the RNA pol by ADP-ribosylation
of the a-CTD.
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Activators CAP
Regulation of the arabinose operon
1
Note that the AraC repressor binding site
overlaps the -35 of the araBAD promoter. AraC
interacts with the a CTD. Stimulates formation of
the closed complex and facilitates formation of
the open complex. CAP serves a similar role in
the araPFGH promoter.
1
ara
2
AraC
2
No glucose arabinose
Johnson Schleif (2000) J. Bact. 182 1995-2000.
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Activators CAP
Regulation of the arabinose operon
AraC
AraC prevents binding at the dead-end site.
No glucose arabinose
Johnson Schleif (2000) J. Bact. 182 1995-2000.
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Activators CAP
Regulation of the arabinose operon
1- At the araBAD and araFGH promoters, both CAP
and AraC are required for activity. 2- At both
promoters, the binding of RNA pol is extremely
tight due to the simultaneous contact of the
a-CTD with AraC and with CAP. 3- AraC only acts
to stimulate when its binding site has the same
orientation as that found in the araBAD
promoter. 4- The a-CTD contacts the AraC protein
at the araFGH promoter.
5- AraC blocks a dead-end site that competes for
RNA pol binding.
Johnson Schleif (2000) J. Bact. 182 1995-2000.