Gene Regulation -- The Lac Operon

While the mechanisms of transcription and translation are still fresh in
our minds, let's take a look at another very significant problem:
specifically, how is gene expression regulated? We know that some genes
are active only at certain times during development and that different
genes are expressed in different tissues. We also know that almost every
cell of a higher organism must have essentially the same set of genes.
This was proven first with plants where a single differentiated phloem cell
of a carrot (and now many cell types from many other plants) could be
induced in culture to regenerate a whole new carrot plant. More recently,
cloning of whole animals has demonstrated the same thing; differentiation
into specific cell and tissue types does discard un-needed genes. Starting
with Dolly, a sheep created when a nucleus from a mammary gland cell
was injected into an enucleated egg, several species of mammals have
been cloned. Examplesinclude a bull Second Chance
www.reporternews.com/1999/texas/clone0902.htmland though not yet
successful, the "Missyplicity" project www.missyplicity.com/to clone a pet dog,
both of which are projects of the TAMU Vet. School. Regeneration of
genetically identical copies, (at least for genes in the nucleus of the
donor) clearly shows that all the genes are still present in mature cells,
even if they are not transcribed in all tissues.

Although it is easy to demonstrate differential gene activity in higher
organisms, it is much simpler to study the molecular events in gene
regulation in simpler organisms. The main effort of this lecture is to
explore an extremely well described system of gene regulation in the
bacterium E. coli,. The system provides a foundation for models that
explain regulation in more complicated systems.

The model we will examine is called the Lac Operon,and it has to do with
the ability of E. colito utilize the sugar lactose. Lactoseis a 12 Carbon
sugar made of 2 simpler 6 Carbon sugars, glucose and galactose. As you
likely know, glucose is a very efficient carbon source; it can enter directly
into the metabolic paths that provide both energy and substrates for
making more complex compounds. If lactose is provided as the carbon
source, it must first be broken down into the two component sugars
before it can be used.

The enzyme for breaking down lactose in E. coli is called β-galactosidase.
The following observations demonstrated that the gene that codes for β-
galactosidase in E. coli is regulated:

E. coligrown in glucose as the sole carbon source have about 3 copies
of the enzyme β-galactosidase/cell.

E. coli grown in lactose as the sole carbon source have about 3,000
copies of the enzyme β-galactosidase/cell.

Thus, there are a thousand times more copies of the enzyme when it is
needed than when it is not.

The system of regulation seen here is called "induction" since synthesis of
the enzyme is "turned on" only when needed. Induction typically is used
to regulate "breakdown" pathways as opposed to "synthetic" pathways.

Two Frenchmen. Francios Jacob and Jaque Monod won a Nobel prize for
their work in describing how the lac-operon functions. They used a
genetic approach to address the problem, by identifying mutants that did
not have normal regulation of β-galactosidase. We will first look at the
model they derived, and then see how the behavior of mutants led to the
model.

The lac-operon is actually a series of adjacent genes and regulatory
elements in one small part of the E. colicircular chromosome.
Lac-Operon components

Definitions:P strands for promoter; it is the site where RNA polymerase
attaches in order to transcribe mRNA.

Although all promoters have the same function and share similar
sequences that are recognized by RNA polymerase, they differ enough so
that some are very strong (leading to high levels of transcription) and
others are weak (rarely transcribed). Thus, one level of regulating gene
expression comes as a consequence of the strength of the promoter at the
beginning of the gene.

P

O

Z

I

Y

A

P

The I gene is called a regulator gene; it is transcribed to make a mRNA
which is translated to a repressor protein. There is a termination signal at
the end of the Igene.

O stands for Operator; it is a short sequence of bases that acts like a
switch that can be recognized by repressor protein.

Z, Yand Aare all "structural genes (genes that code for polypeptides)

Z codes for β-galactosidase; Y codes for lactose permease, a protein that
functions to actively bring lactose from outside to cell to the inside, even
against a concentration gradient. A codes for transacetylase, an enzyme
that is also needed to breakdown many sugars related to lactose.

One long mRNA is made for the Z, Y and A genes; this is the basis for the
system being called an operon. All 3 genes that code for enzymes needed
to use β-galactoside molecules as a source of carbon and energy are
adjacent and are coordinately turned on or off by regulating
transcription. Operons are only found in prokaryotes; in eukaryotes, each
structural gene has its own promoter and regulatory elements.

Lets first examine how the lac-operon functions when only glucose is
present; that is when we expect it to be turned off:

Stepwise: 1. The Promoter for the I gene is always "on", but is very weak,
so it is transcribed only rarely. A gene that is not regulated,

P

P

O

Z

I

Y

A

transciption

mRNA

repressor protein

translation

1

2

3

other than by the strength of its promoter, is said to be
"constitutive".

2. The I mRNA is translated into the repressor protein. A
typical cell will have only about 10 copies of this protein.

3. In the absence of lactose, the repressor protein binds to the
Operator, preventing transcription form the second
promoter. Almost no ZYA mRNA is made.

When only lactose is present the model works as follows:

Stepwise: 1. The Promoter for the I gene occasionally is bound by an
RNA polymerase to initiate transcription.

2. The I mRNA is translated into the repressor protein; a typical
cell will have only about 10 copies of this protein.

3. Lactose (actually one stereo-isomer called allolactose) binds
to the repressor very efficiently and converts the repressor
into an inactive state, where it can't bind the Operator. The
process is reversed when all the lactose is digested, so the
system again will turn off.

4. When the very strong Promoter for making Z-Y-A mRNA is
not blocked, many copies of the mRNA are made. The small
amount of lactose that diffuses in is able to initiate induction

transciption

mRNA

repressor protein

translation

1

2

3

P

O

Z

I

Y

A

P

lactose⁽⁾

inactive repressor

mRNA

b-gal'ase

permease

TA'ase

4

5

of transcription of the Z-Y-A mRNA. Even as the message is
being made, translation begins and the 3 proteins are made.

5. Translation begins at the 5' end of the mRNA and makes β-
galactosidase from the Z gene. There is a stop codon,
followed immediately by another AUG start, so many, but not
all, ribosomes read on through and make permease from the
Y gene. The same process allows some A gene product to
also be made.

This whole system of regulating gene expression by regulating
transcription is especially effective because messages are the most
fragile part of the system. The average half-life of a mRNA in E. coli
is about 1.8 minutes. Messages in eukaryotic tissues have a longer
"life expectancy", but on the average are still much more fragile than
proteins. It should be pointed out though that even proteins "turn-
over", that is they wear out or become nonfunctional and are subject
to degredation.

(continued on the next page)

Mutations that define the Lac-Operon model:

Jacob and Monod found mutants that did not show the normal regulation,
that is, synthesis of Z, Y, and A proteins only when lactose was present.

Two kinds of mutants gave constitutive (continuous) synthesis of β-
galactosidase, permease and TAase, no matter what carbon source was
present

a) Mutants in the I gene(i^-or i^c) mutants all were mapped (we will
take up mapping later in the course) to a similar location. Special
tricks were used to make cells that were partial diploids with two
copies of the lac-operon. When the copies created heterozyous cells

(I , I^-) normal regulation was observed. This indicated that the I gene
codes for something that can move and interact with the operators of
both copies of the lac-operon present in these cells.

+

b) Mutants in the promoter that change the base sequence so that it is
no longer recognized by the repressor protein are also constitutive for
Z, Y and A expression. In this case however, the mutation is dominant
in partial diploids:

mRNA

repressor protein

P

O

Z

I

Y

A

P

O

Z

i^-

Y

A

P

P

Since the operator in the upper strand cannot be bound by active
repressor, β-galactosidase and TAase will always be made. In this cell,
permease will show normal regulation since it is only made by the
lower copy of the lac-operon. These results told Jacob and Monod that
the Operator regulated transcription only of gene on the same DNA
molecule.

From this simple model it is easy to see how a cascade of events could
lead to complex regulatory schemes; the product of one reaction could
induce the next pathway etc. until development is accomplished.

Analogous systems exist in bacteria for regulation of biosynthetic
pathways. For example, if we look at the pathway for the synthesis of the
amino acid histidine, several levels of regulation can be seen. Since E. coli
spend part of their life cycle in a colon, they may well have plenty of
histidine available from digested proteins. On the other hand, if no
histidine is available they can make it "from scratch". The pathway for
histidine biosynthesis starts with ATP+ PRPP and after 11 enzyme
catalyzed reaction histidine is made. Given that a bacterium that wastes
the resources and energy used to make histidine when it is not needed

transciption

mRNA

repressor protein

translation

1

2

P

O

Z^-

I

Y

A

P

mRNA

TA'ase

4

P

O^c

Z

I

Y^-

A

P

mRNA

β-gal'ase

would not compete well in nature, it is not surprising that several levels of
regulation has evolved. One very common mechanism for biosynthetic
(anabolic) pathways that is used for fine-tuning the level of the product is
called "end-product inhibition". In end-product inhibition, the product of
the pathway binds to the first enzyme unique to the pathway to inactivate
it, thus slowing the rate of synthesis. As is the case with lactose
interaction with the lac-repressor, histidine does not bind to the active
site of the enzyme but to a different (allosteric site) which causes the
protein to assume an inactive configuration. The reaction is also
reversible, so that when the endproduct concentration becomes low, the
enzyme will regain its active conformation.

However, if there is really an excess of histidine, it would also be costly to
make 11 unneeded enzymes only to shut off the first enzyme. In this
case, a systemmuch like the lac-operon takes over. In his case, since the
function is to turn off genes when a compound is present, the model is
an example of repression.

PReg

POE1E2E3E4E5 E6 E7E8E9E10E11

In the case of the histidine operon, the regulator gene product is called a
co-repressor. By itself, it cannot bind to the Operator unless it is first
bound to histidine. Thus, a relatively subtle difference accounts for
regulation in the inducible lac-operon and the repressible his- operon.

Many other variations on the same theme are known. For example, if
both glucose and lactose are present cells use up the glucose before
turning on the lac-operon. When energy begins to become limiting, a
signal molecule (cAMP) builds up, binds to a CAP protein, which in
turn binds to a site between I and P
₂of the lac operon, which opens the
promoter for RNA polymerase binding.

co-repressor

histidine

active co-
repressor