Transcript 1. Translation

1. The logic of prokaryotic transcriptional regulation
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In addition to the sigma factors that allow RNA polymerase to bind
the promoter, another type of DNA-protein interaction regulates
whether or not promoter-driven transcription occurs.
DNA segments near the promoter serve as protein-binding sites for
regulatory proteins called activators and repressors; these sites on
DNA are termed operators.
For some genes, the binding
of an activator protein to its
target DNA site is a necessary
prerequisite for transcription
to begin (positive regulation).
For other genes, preventing
the binding of a repressor
protein to its target site is a
necessary prerequisite for
transcription to begin
(negative regulation).
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
2. Activators, repressors, effectors
For activator or repressor proteins to do their job, each must be able to
exist in two states: one that can bind its DNA targets and one that cannot.
The binding state must be in accord with the cellular environment; that
is, be appropriate for a given set of physiological conditions.
A site on the regulator protein interacts with
small molecules called allosteric effectors;
these act as toggle switches that sets the
DNA-binding domain in one of two modes:
functional or nonfunctional. An allosteric
effector binds to the allosteric site of the
regulatory protein in such a way that it
changes the structure of the DNA-binding
domain. Some activator or repressor proteins
must bind to their allosteric effectors to bind
DNA. Others can bind DNA only in the
absence of their allosteric effectors.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
3. A molecular switch
DNA-bound activator proteins act at
the level of transcription initiation, by
physically helping to bind RNA
polymerase to its nearby promoter. A
DNA-bound repressor protein
typically acts either by physically
interfering with the binding of RNA
polymerase to its promoter (blocking
transcription initiation) or by
impeding the movement of RNA
polymerase along the DNA chain
(blocking transcription elongation).
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
4. Regulation of the Lactose System
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A relevant example of transcriptional regulation in prokaryotes is the
control of the enzymes necessary for lactose metabolism in E. coli.
Most of the models and mechanisms involved in this specific system
have been revealed by genetic analyses of mutated bacterial strains.
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Presumably because of energy-efficiency considerations, two environmental
conditions have to be satisfied for the lactose metabolic enzymes to be
expressed.
One condition is that lactose must be present in the environment. It would be
inefficient for the cell to produce the lactose metabolic enzymes in
circumstances where there is no substrate to metabolize.
The other condition is that glucose should not be present in the cell's
environment. Because glucose metabolism yields more usable energy to the cell
than does lactose metabolism, mechanisms have evolved that prevent the
synthesis of the enzymes for lactose metabolism in the presence of glucose.
Genetica per Scienze Naturali
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5. Introducing the operon
A simplified lac operon model. The three genes Z, Y, and A are
coordinately expressed. The product of the I gene, the repressor, blocks
the expression of the Z, Y, and A genes by interacting with the operator
(O). The inducer can inactivate the repressor, thereby preventing
interaction with the operator. When this happens, the operon is fully
expressed.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
6. The metabolism of lactose
The metabolism of lactose requires two enzymes: a permease to transport lactose into
the cell and b-galactosidase to cleave the lactose molecule to yield glucose and
galactose (Figure 14-4). Permease and b-galactosidase are encoded by two contiguous
genes, Z and Y, respectively. A third gene, the A gene, encodes an additional enzyme,
termed transacetylase, but this enzyme is not required for lactose metabolism.
All three genes are transcribed
into a single, multigenic
messenger RNA (mRNA)
molecule. Regulation of the
production of this mRNA
coordinates the regulation of the
synthesis of all three enzymes.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
7. The gene for the Lac repressor
and the lac operator site
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A fourth gene, the I gene, encodes the Lac repressor protein,
so named because it can block the expression of the Z, Y,
and A genes. The I gene happens to map fairly near the Z, Y,
and A genes, but this proximity does not seem to be
important to its function.
The operator (O) is the site on the DNA to which the Lac
repressor binds. It is located between the promoter and the Z
gene near the point at which transcription of the multigenic
mRNA begins.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
8. The lac operon is regulated by lactose
The P, O, Z, Y, and A segments constitute an operon, which is a genetic unit of
coordinate expression. The interaction between the lac operator site on the DNA and the
Lac repressor is crucial to proper regulation of the lac operon. The Lac repressor is a
molecule with two recognition sites, a DNA-binding site that can recognize the specific
operator DNA sequence for the lac operon and an allosteric site that binds the lactose
allosteric effector and similar molecules (analogs of lactose).
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
9. The lac repressor
Lac repressor is a tetrameric protein organized as a dimer of dimers.
Each component homodimer forms one DNA binding region from two
equivalent chains. The lac operator sequence is an almost perfect
palyndrome, recognized by inserting one helix-turn-helix motif from
each chain of the lac repressor dimer into the DNA major groove of the
half palyndrome.
This helix-turn-helix motif is found to be
common to a variety of bacterial and phage
repressor or DNA binding proteins
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
10. The complex between the lac repressor
and the lac operator
The DNA-binding site of the Lac repressor is able to bind with high affinity to only one
DNA sequence in the entire E. coli genome, the lac operator. The specificity of highaffinity DNA binding ensures that the repressor will bind only to the site on the DNA
near the genes that it is controlling and not to random sites distributed throughout the
chromosome. By binding to the operator, the repressor prevents transcription by RNA
polymerase that has bound to its lac promoter site
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
11. A second mechanism of control
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In the absence of lactose, the Lac repressor binds to the lac operator site and
prevents transcription of the lac operon, by blocking the progression of RNA
polymerase transcription.
Consequently, all of the structural genes of the lac operon (the Z, Y, and A genes) are
repressed, and there is no b-galactosidase, b-galactoside permease, or transacetylase
in the cell.
In contrast, when lactose is present, it binds to the allosteric site of the Lac repressor,
thereby inactivating the operator DNA-binding site of the Lac repressor protein.
This inactivation permits the induction of transcription of the structural genes of the
lac operon and, through the translation of the multigenic mRNA, the enzymes bgalactosidase, b-galactoside permease, and transacetylase now appear in the cell in a
coordinated fashion
However, there is more to the regulation of lac operon transcription. The above
mechanism satisfy only one of the conditions that the lac operon should obey; the
entire system also requires a second environmental condition, namely, that glucose
is not present in the environment of the cell.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
12. The effect of glucose
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An additional control system is superimposed on the repressor
operator system. This control system is thought to have evolved
because the cell can capture more energy from the breakdown of
glucose than it can from the breakdown of other sugars.
If both lactose and glucose are present, the synthesis of bgalactosidase is not induced until all the glucose has been utilized.
Thus, the cell conserves its energy pool used, for example, to
synthesize the Lac enzymes by utilizing any existing glucose before
going through the energy-expensive process of creating new
machinery to metabolize lactose.
It is a breakdown product of glucose (the identity of this catabolite is
as yet unknown) that prevents activation of the lac operon by lactose;
this is the catabolite repression mechanism.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
13. cAMP and CAP
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A glucose catabolite modulates the level of an important cellular
constituent, cyclic adenosine monophosphate (cAMP).
When glucose is present in high concentrations, the cell's cAMP
concentration is low; as the glucose concentration decreases, the
cellular concentration of cAMP increases correspondingly.
The high concentration of cAMP is necessary for activation of the lac
operon.
cAMP is an effector of a protein, called CAP (catabolite activator
protein), which is coded by the crp gene. In absence of cAMP , CAP
cannot bind to the CAP site, while bound to cAMP, CAP is able to
bind to the CAP site.
The DNA-bound CAP is then able to interact physically with RNA
polymerase and essentially increase the affinity of RNA polymerase
for the lac promoter. CAP is an activator protein.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
14. CAP binding to DNA-CAP binding site
The CAP protein active sites are modified by
the presence of cAMP so that the complex
binds at the proper DNA site in the promoter
of the lac operon.
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Genetica per Scienze Naturali
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15. The 5’ lac operator control region
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16. The effect of cAMP-ligated CAP
Genetica per Scienze Naturali
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17. A summary of the lac operon control
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini