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BCH 415 Note: The Lactose Operon—A Look at Regulation
The biochemistry of the lactose ( lac) operon explains many principles of regulation
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Lactose is a
disaccharide composed of two sugars (galactose and glucose) with a β-linkage between carbon 1 of
galactose and carbon 4 of glucose, as shown in Figure 1
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The function of
\cA is not known, but a mutation in either lacZ or lacY means that the cell can't grow by using lactose as a
sole carbon source
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The lac transcript is termed polycistronic because it contains more than one coding
sequence
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The repressor has two functions
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Secondly, it binds to a small molecule called an inducer
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The binding of inducer to the repressor is
cooperative, meaning that the binding of one molecule of inducer makes binding of the next one more
favorable, and so on
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In the absence of inducer, the repressor protein binds to a sequence called the operator ( lacO) which
partially overlaps with the promoter
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Repressor is
therefore a negative regulator of gene expression: If repressor is not present (for example, the bacterium is
deleted for the lacI gene), then transcription of the lac genes occurs, and the structural genes are expressed
whether or not inducer is present
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This behavior is characteristic of a negative control element
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Deletion of lacO leads to constitutive expression of the lac genes
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See Figure 2
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This behavior is characteristic of a diffusible control element, and the repressor is said to act in
trans
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This is characteristic of a site where the product of another gene
acts, and lacO is therefore termed cis-acting
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If the
repressor can't bind inducer, then the lac genes it controls are permanently turned off because repressor
will be bound to the operator whether inducer is present or not
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A second level of control is superimposed on the repressor-operator interaction described previously
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This makes good metabolic sense
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The phenomenon by which
glucose reduces the expression of the lac operon is called catabolite repression, as shown in Figure 3
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The first component is the small-molecule regulator, cyclic
AMP
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The second component is cyclic AMP binding protein,
CAP
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When bound to cAMP,
CAP binds to a sequence at the 5′ end of the lac promoter
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It therefore behaves in the opposite manner of repressor
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These two complementary systems allow the bacterial cell to metabolize lactose in response to two stimuli
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This series of switches allows complex expression patterns to be built up from simple components
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Corepressors
As mentioned above, the synthesis of tryptophan from precursors available in the cell requires 5 enzymes
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In
this case, however, the presence of tryptophan in the cell shuts down the operon
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When Trp is not
present, the repressor leaves its operator, and transcription of the 5 structural genes begins
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The presence in the cell of an essential
metabolite, in this case tryptophan, turns off its own manufacture and thus stops unneeded protein
synthesis
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However, some gene transcription in E
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Positive Control of Transcription: CAP
Absence of the lac repressor is essential but not sufficient for effective transcription of the lac operon
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Like the lac repressor, CAP has two types of binding sites:




One binds the nucleotide cyclic AMP; the other
binds a sequence of 16 base pairs upstream of the promoter

However, CAP can bind to DNA only when cAMP is bound to CAP
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So the lac operon is under both negative (the repressor) and positive (CAP) control
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This dual system enables the cell to make
choices
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coli (for reasons about which we can only speculate) chooses glucose
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Without CAP, binding of RNA polymerase is inhibited even though there is no repressor to interfere with
it if it could bind
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CAP consists of two identical polypeptides (hence it is a homodimer)
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The longer of these is called the recognition
helix because it is responsible for recognizing and binding to a particular sequence of bases in DNA
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The two monomers are identical
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Note that the two
recognition helices are spaced 34Å apart, which is the distance that it takes the DNA molecule (on the left)
to make precisely one complete turn
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But their orientation
in the dimer is such that the sequence of bases they
recognize must run in the opposite direction for
each recognition helix to bind properly
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The strategy illustrated by CAP and its binding site has turned out to be used widely
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Therefore, they are dimers
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In bacteria, recognition and binding to a particular sequence of DNA is accomplished by a segment
of alpha helix
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The Trp
repressor shown above is a member of this group
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It
turns out that the regulation of the level of certain metabolites can also be controlled by riboswitches
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Some of the metabolites that bind to riboswitches:
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the purines adenine and guanine
the amino acids glycine and lysine
flavin mononucleotide (the prosthetic group of NADH dehydrogenase)
S-adenosyl methionine (that donates methyl groups to many molecules, including
o DNA
o the cap at the 5' end of messenger RNA

In each case, the riboswitch regulates transcription of genes involved in the metabolism of that molecule
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In both cases, one result is to control the level of that metabolite (a kind of feedback inhibition)
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It has been suggested that these regulatory mechanisms, which do not involve any protein, are a relict
from an "RNA world"

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