6. CAP
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Lac Book 3.2-Some numbers
•Muller-Hill has an on going fascination with numbers and biology. In
particular, he has a long-standing interesting in trying to think about
biological phenomena in terms of how they are affected by how many
molecules of any protein are in a cell, their concentrations, how often they
bump into each other etc.
Numbers pertaining to E. coli
•typical cell is 2um by 1um
•volume is 2 x 10-12 ml
•if a cell contains 1 molecule of something, the concentration of that
something is 1x10-9 M. (What does this assume? That the whole cytoplasm
is water, and available for the something to be dissolved in.) Water makes
up ~1/3 of the cytoplasmic volume.
•The lacZ gene is 1um long
•E. coli has ~4.5 million base pairs of DNA. A sequence must therefore be
about 11 bp long in order to be unique (on average). 411= ~4x106. (? How
often should one find TTACT in the E. coli chromosome, assuming the
genome has equal amounts of all 4 bases?)
•One cell has 3,000 RNA polymerase molecules.
•One cell has 10 copies of tetrameric Lac repressor (~10-8 M)
•An average cell has 1.7 copies of lac operon when chromosomes are
replicating.
•When fully induced, cells will inititate transcription from plac once every
1.7 seconds.
•RNAP transcribes at 80 bases per second
•The lacZ gene is 3000 basepairs long
•Therefore, a newly initated RNAP travels 1.7x80=136 bp before the next
RNAP initiates. This means that lacZ has 3000/136 = 22 RNAP molecules
transcribing it on average (plus one at the promoter).
•It takes about 37 seconds to transcribe lacZ, and once fully induced a new
lacZ mRNA will be made every 1.7 seconds.
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Lac Book 3.3 Activation of plac by CAP
3.3.1 Modular structure of CAP
•Binds to DNA in the presence of cAMP, but not in the absence of cAMP
(slide)
Muller-Hill
states
and
others
thought
that
CAP dimer with one bound cAMP bound DNA best (seen at 100 uM) and
that at 1mM 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. (slide)
•CAP dimer has HTH domains that are spaced properly to fit together into
the major groove of DNA, unfortunately, they are not in the proper
orientation to do so. So, Anderson and Steitz proposed that CAP bound
left handed DNA, not the more common right-handed DNA, they even
tried re-orienting the HTH to make it more likely that it would bind right
handed DNA, but to no avail. (slides)
•10 years later Steitz, solved the structure of CAP bound to DNA and
found that the DNA was in the right-handed configuration (not lefthanded as he thought), but it was bent by ~90o by the action of two 40o
bends (slide).
•cAMP and diauxie: (Muller-Hill mentions "embarassing news"--> cAMP
levels are the same on glucose and lactose cites Inada et al)
Inada et al show: (Slides)
-levels of cAMP are the same on glucose and lactose
-cAMP spikes as glucose is depleted
-addition of cAMP alleviates diauxie, but does not stop glucose
repression of b-gal expression
•Q: If b-gal is not made when glucose is present, even when cAMP is
added, then what is keeping it off (it’s not low cAMP!)?
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•A: lacZ is kept off because glucose transport inhibits transport of lactose.
-An aside: How is glucose transported into E. coli?
-By a system different, and more complex, than LacY or the typical
PBP, ABC transporters like the Agp system of S. meliloti.
The PTS is made of 2 main parts:
Part 1: Enzyme EI (ptsI), Hpr (ptsH), EIIAglc(crr): operon: ptsH, I, crr
Soluble proteins-->phosphotransfer
Part 2: Enzyme EIIB, EIIC: found together in one fused gene ptsG
Membrane protein with channel and phosphotransfer function
•The glycolytic metabolite PEP phosphorlates EI which phosphorylates
Hpr, which phosphorylates EIIAglc, which phosphorylates EIIB which
phosphorylates glucose when it comes through the channel made by EIIC.
(Slides)
•So what does cAMP do? (slide)
•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 which allows quick inducer concentration,
allowing a shortened lag time
3.3.2 Mutational analysis of the lac promoter and CAP site
•The lac promoter is suboptimal esp in the -10 region.
TTGACA N17 TATAAT vs TTTACA N17 TATGTT
consensus
plac
•This suboptimal sequence transcribes much better (~50x) when
CAP•cAMP binds upstream and interacts with the -subunits of RNAP.
•Mutating the the -10 region to TATAAT makes the promoter much better
and it no longer requires CAP or cAMP for high level expression. This is
the famous lacUV5 promoter mutation
•The CAP binding site itself is far from optimal. Richard Ebright figured
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out the optimal CAP binding site consensus and showed that it bound
CAP about 450x better than the site at the lac promoter region.
Muller-Hill points out that even a large increase in binding affinity would
really only increase the rate of transcription by a small amount: less than 2x.
A. Kd for CAP binding to lac promoter: about 10 nm
(Ebright_NAR_1989).
B. There are ~1300 CAP molecules per cell (~650 dimers) and ~250
binding sites (Zheng_NAR_2004). So if all CAP sites except lac's were
bound there would still be ~400 dimers left.
C. This would be ~400 nM about 40x what is needed to bind the CAP site
at the lac promoter, assuming cAMP is prevalent.
D. So increasing the ability of the lac CAP site to better bind cAMP is not
really needed
3.3.3 Mutational analysis of the CAP protein
•Explain supression and supressor mutations
Definition: The fixing or lessening of a mutant phenotype by a
second mutation at a second site.
•Nonsense suppressors were one example of this. There are others
(see slides)
•Hiroji Aiba took a strain unable to make cAMP and without the CAP
gene, and put into it CAP genes which had been randomly mutated. These
cells he plated onto lactose indicator plates and looked for Lac+ colonies. -He was looking for CAP mutants where the CAP protein activated
transcription at lac, but no longer needed cAMP to do so (aka crp*) (he was
looking for CAP mutations which supressed the cAMP mutation.). He
found 5 mutants that were altered at codons 53, 62, 141, 142 and 148.
•Sankar Adhya did a similar experiment and found suppressors at CAP
codons 72, 141, 142 and 144.
Where do these map? (slides). These sites map around the cAMP binding
site and around a hinge region that allows the DNA binding domain swing
into position when cAMP binds.
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So, the supressor mutations either act to shift the CAP protein into
its cAMP-bound conformation, or to stabilize the form where the DNAbinding domain is in position to bind DNA even though cAMP is absent.
Transcriptional activation by CAP
Transcription
There is one RNA polymerase in bacteria responsible for mRNA, tRNA
and rRNA synthesis. It is not the same as the one that is involved in
priming DNA synthesis.
Promoter structure
•Two parts -35 region and the -10 region
-For housekeeping genes the promoter is similar to:
TTGACA-N14-TATAAT
•These regions are -35 bp and -10 bp to the left of the start of the mRNA
which begins at a site called the +1 site.
Determination of important binding sites by finding consensus
sequences
RNAP composition:
2 x  subunit (gene: xx)--These bind regulatory sequences near
promoters
2 x subunit (rpoA)-binds DNA
1 x  subunit (rpoB)- binds ribonucleotides
1 x ’ subunit (rpoC)-binds DNA
1 x  subunit-rpoZ-RNAP assembly and transc. control
1 x  subunit (rpoD, rpoH, rpoS and others)-binds -35 and -10
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Sigma Factors
•RNAP binds to promoters via interaction of the  subunit with the -35
and -10 boxes. Sigma factors are elongated proteins that contact the -35
region with their d4 domain and the -10 region with the d2 (d2.4) domain.
(SLIDES- RNAP and Sigma)
LacBook 3.3.4 pg 151
•The simplest hypothesis concerning CAP was that it bound to DNA and
helped RNAP bind to poor promoters. This was tested by Richard Ebright.
He made a 42 bp DNA that bound CAP and had no promoter region and
therefore did not bind RNAP.
However, when w.t. CAP was added RNAP was able to bind by
interacting with CAP that was bound to the DNA
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When a mutant CAP which bound DNA, but did not activate
transcription was added, RNAP did not bind showning that RNAP did in
fact bind to CAP(wt) (SLIDES from Heyduck_Nature_1993)
•Igarashi and Ishihama (Igarashi_Cell_1991) showed that an rpoA mutant
missing the last 73 aa of RNAP a-subunit could transcribe from good
promoters, but not from ones which required CAP for activation. This
suggested that the last part of a-subunit is needed for RNAP to contact
CAP. Later, Ebright's lab showed that point mutations in the C-terminus
of rpoA had the same effect (Batter_Cell_1995)
Note: not all CAP-RNAP interactions are via alpha-subunit (those that are
are called "Class I" and are upstream of the promoter) . Sometimes the
CAP site is overlaps the -35 site and binds RNAP at multiple sites
including the Cterm of alpha.(Slides showing mechanisms of
transcriptional activation)
Global control of expression by CAP
In order to see what genes are controlled by CAP on a global scale, Zheng
et al. (2004) did a ROMA experiment (run off microarray analysis).
Data from this experiment is shown in Zheng_NAR_2004 Fig 1(slide)
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The experiment was also done with no CAP•cAMP and CAP mutant
protein that couldn't do type 1 activation (CRP HL159) and one that
couldnt do type 2 activation (CRP KE101). Both of these experiments
show that there are genes that are regulated by either type1 or type2
activation (Slide)