Only CAP – cAMP complex binds to the lac promoter ; in the absence of cAMP, CAP

does not bind. Thus cAMP as the efector molecule, determining the effect of CAP on lac operon
transcription. The intacellular cAMP concentration is sensitive to the precense or absence of
glucose. High concentrations of glucose cause sharp decreases in the intracellular concentration
of cAMP. How glucose controls the cAMP concentration is not clear. Perhaps glucose, or some
metabolite that forms in the presence of suffleient concentrations of glucose, inhibits the
activity of adenylcyclase, the enzyme that catalyzes the formation of cAMP from ATP.
Whatever the mechanism, the presence of glucose results in the decrease in the intracellular
concentrations of cAMP. In the absence of (or in the presence of the low concentration of)
cAMP, CAP cannot bind to the lac operon promoter. In turn, RNA polymerase cannot bind
effeciently to the lac promoter in the absence of bound CAP. The overall result of the positive
control of transcription of the lac operon by the CAP-cAMP complex is hat in the presence of
glucose, lac operon trancrition never exceeds 2 percent of the induced rates observed in the
absence of the glucose.

The complete nucleotide-pair sequence of the lac operon regulatory (the promoter and
operator sequnces) is now know (fig 14.7). Comparative nucleotide sequence studies of mutant
and wild type promoters and operators (plus in vitro CAP-cAMP, beginning to provide detailed
information about the nature of these important sequence-specific protein nucleid acid
interactions. These studies provide an excellent example of the advantages of integrating
genetic and biochemical approaches in attempting to understand biological phenomena.

Complex Regulation of the ara Operon

Almost the details about the mechanisms by which the lac and trp operons are regulated
are known and supported by an extensive body of experimental data. However, other operons
ike the arabinose (ara) operon of E.coli exhibit much more complex patterns of the regulation
that are still not completely understood. In the lac and trp operons the product of the regulator
gene, the repressor, funtions in the negative manner, turning off trancription of the operon. On
the other hand, the catabolite activator protein (CAP) exerts a positive control over the lac
operon by stimulating trancription of the operon. The major regulatory protein of the ara operon
exhibits both negative and positive regulatory effects on the transcription of the structural genes
of the operon depending on the environmental conditions. Moreover, the regulatory
components that control trancription of the ara operon include one element that acts from a
distance of over 200 nucleotide-pairs away from the promoter that operon regulatory circuitry
are not yet established unambiguously, the current working model is precented here to give the
student an appreciation for the complexity of the regulation of come bacterial operons.

The arabinose (ara) operons of the E.coli contains three structural (araB,araA, and
AaraD) that encode the three enzymes inolved in the catabolism of arabinose. (fig 14.8a). These
three genes are contranssribed on the single mRNA that is initiated at a promoter called PBAD
(active transport of arabinose into the cells is carried out by the products of genes araE,araF,
and araG. These genes are located at sites quite distant from the araBAD operon of interest
here and protein of the ara operon (the araC protein) is produced from transcript that is initiated
at a promoter called Pc. The Pc promoter is only slightly over 100 nucleotide-pairs away from
PBAD but the two promoters initiate transcription in opposite directions (14.8a and b). The araC
protein acts as negative regulator (a repressor) of transcription of the araB,araA, and araD
structural genes from the PBAD promoter when arabinose and cAMP are present. Thus,
depending on the presence or absence of the effector molecules arabinose and cAMP, the araC
regulatory gene product may exert either a positive or a negative effect on transcription of the
araB,araA and araD structural genes. Since ara operon is subject to catabolite reppression like
the lac operon (see the precending section) and thus to positive control by CAP and cAMP,
induction of ara operon depends on the positive regulatory effects, of two proteins, the araC
protein and CAP (the cAMP binding catabolite activator protein). The binding sites for these
two proteins and for RNA polymerase all appear to be located in a region of the ara operon
historically called araI (I for induction).,located between the three structural genes of the
operon and the regulator gene (araC) (fig 14.8a)

Initially,scientist studying the regulation of the ara operon thought that all the binding
sites for the araC regulatory protein and the cAMP-CAP complex were in the aral region. The
surprising discovery was that repression on of the ara operon depended on the binding of araC
proteion at a site called araO2 (O for operator, 2 because it was the second ara operator
identified) located 211 nucleotide-pairs upstream (relative to the direction of trancription from
PBAD ) from the araC protein-binding site in araI (fig 14.8b). (Operator araD I- the first ara
operator to be indentified-controls the transcription of the araC regulator gene initiated at Pc).
The currently accepted model for the repression of the ara operon is that the araC protein must
bind (as dimer) at both the araI site and the araO2 site, and that these proteins then bind to each
other to form a DNA loop (fig 14.8c). In fact, there is now considerable evidence in support of
this model. For example, if five nucleotide-pairs are inserted or deleted in the region between
araI and araO2, normal repression of the operon cannot occur. Such an insertion or deletion will
rotate one araC cprotein binding-site halfway around the double helix(to the opposite face)
relative to the other araC protein-binding site. This presumably would make it difficult or
impossible for the araC dimers bound at araI and araO2 to interact and form the loop required
for repression.

When the loop structure (fig 14.8c) is formd, it must present or interface with the
binding of RNA polymerase at the adjacent promoter (PBAD) of the operon (fig 14.8b) in the
presence of arabinose and cAMP, the ara operon is induced. Moreover, under these conditions,
the araC protein has been shown to become an activator of transcription of the operon. The
details of the mechanism by which arabinose causes the araC protein to become a positive
regulator of transcription of the operon are not clear. Somehow, the arabinose-araC protein
complex and the cAMP-CAP complex must open the loop by binding at their araI sites. This,
in turn, must permit RNA polymerase to bind at the PBAD site and initiate transcription of the
ara structural genes (fig 14.8d).

Clearly, the regulation of transcription of the ara operon E.coli is considerably more
complex than the regulation of transcription of the lac operon of this bacterium. It will be
interesting to etermine whether or not this loop formation mechanism is commonly employed
in the regulation of transcription of other operon in prokaryotes or of genes in eukaryotes.

Lambda Prophage Repression During Lysogeny

When a temperate bacteriophage sush as lambda exists in the prophage state in the
lysogenic cell, the genes coding for products involved in the lytic pathway-namely, the genes
controlling phage DNA replication, phage morphogenesis, and lysis of the host cell-must not
be expressed. This is accomplished by a repressor-operator-promoter circuit, much like that
involved in bacterial operons. Specifically, the C1 gene of phage lambda codes for repressor,
a well characterized protein with a molecular weight 27.000, which in the dimer or tetramer
state binds to two operator regions that control transcription of the lambda gene involved in
lytic growth (fig.14.9). these two operator region, termed OL (for transcription in a leftward
direction) and OR ( for transcription in the rigtward direction), overlap with promoter sequences
as which RNA polymerase binds and initiates transcription of the genes conrolling lytic
development. Thus with the repressor bound to the two operators, RNA polymerase cannot
bind to the two promoters and cannot, therefore initiate transcription. In this way the phage
genes are kept repressed, allowing the “dormant” prophage to be transmitted from parental host
cells to progeny cells generation after generation.

In experiments in which the operator and promoter regions of phage lambda were
sequenced, each operator was unexpectedly found to contain three repressor binding sites with
similar, but no identical, sequences of 17 nucleotide-pairs. Each repressor binding site has
partial twofold symmetry around the central base pair (i.e.,is partially palindromic). It has been
speculated that this partial symmetry many fasilitate interaction with repressor dimers, which
might also have twofold symmetry. Although this is an attractive posibility, it is nothing more
than that at present.

The interaction of lambda repressor with DNA sequences O LPL and ORPR nicely
explains how the lambda prophage genes are maintained in the repressed state. The mechanism
responsible for the decision between lytic development and lysogenic development after
infection of an E.coli cell with a lambda phage is considerably more complex, involving
interctions among several other lambda regulatory genes. The reader is reffered to one of the
papers by Ptashne and cowokers for a discussion of the mechanism by which the choice
between the lytic pathway an =d the lysogenic pathway is made.

CONTROL OF THE trp OPERON BY ATTENUATION

Repression and derepression can change the level of the expression of the structural genes og
the trp operon by about 70-fold. There is a second level of regulation of trp operon expression,
however in trpR mutants that cannot make repressor, the addition of tryptophan to a culture of
cells growing