Regulation of the 

lac Operon

The activity of the promoter that controls the expression of the lac operon is

regulated by two different proteins. One of the proteins prevents the RNA
polymerase from transcribing (negative control), the other enhances the binding of
RNA polymerase to the promoter (positive control).

Negative Control of the lac Operon

The protein that inhibits transcription of the lac operon is a tetramer with four

identical subunits called lac repressor. The lac repressor is encoded by
the lacI gene, located upstream of the lac operon and has its own promoter.
Expression of the lacI gene is not regulated and very low levels of the lac repressor
are continuously synthesized. Genes whose expression is not regulated are called
constitutive genes. In the absence of lactose the lac repressor blocks the expression
of the lac operon by binding to the DNA at a site, called the operator that is
downstream of the promoter and upstream of the transcriptional initiation site. The
operator consists of a specific nucleotide sequence that is recognized by the
repressor which binds very tightly, physically blocking (strangling) the initiation of
transcription. The lac repressor has a high affinity for lactose. When a small
amount of lactose is present the lac repressor will bind it causing dissociation from
the DNA operator thus freeing the operon for gene expression. Substrates that
cause repressors to dissociate from their operators are called inducers and the genes
that are regulated by such repressors are called inducible genes.

Positive Control of the lac Operon

Although lactose can induce the expression of lac operon, the level of expression is
very low. The reason for this is that the lac operon is subject to catabolite
repression or the reduced expression of genes brought on by growth in the presence
of glucose. Glucose is very easily metabolized so is the preferred fuel source over
lactose, hence it makes sense to prevent expression of lac operon when glucose is
present.

The strength of a promoter is determined by its ability to bind RNA polymerase

and to form an open complex. The promoter for the lac operon is weak and
consequently the lac operon is poorly transcribed upon induction. There is a
binding site, upstream from the promoter, for a protein called the catabolite
activator protein (CAP). When the CAP protein binds it distorts the DNA so that
the RNA polymerase can bind more effectively, thus transcription of
the lac operon is greatly enhanced. In order to bind the CAP must first bind cyclic
AMP (cAMP), a second messenger synthesized from ATP by the enzyme
Adenylate Cyclase. In the presence of glucose circulating cAMP levels are very
low and consequently the initiation of transcription from the lac operon is very
low. As glucose levels decrease the concentration of cAMP increases activating
CAP which in turn binds to the CAP site stimulating transcription. The cAMP-
CAP complex is called a positive regulator.

The Tryptophan Operon

E. coli can synthesize all 20 of the natural amino acids. Amino acid synthesis
consumes a lot of energy, so to avoid wasting energy the operons that encode for
amino acid synthesis are tightly regulated. The trp operon consists of five
genes, trpE, trpD, trpC, trpB and trpA, that encode for the enzymes required for
the synthesis of tryptophan. The trp operon is regulated by two mechanisms,
negative corepression and attenuation. Most of the operons involved in amino acid
synthesis are regulated by these two mechanisms.

Negative Corepression

The trp operon is negatively controlled by the trp repressor, a product of

the trpR gene. The trp repressor binds to the operator and blocks transcription of
the operon. However, in order to bind to the operator the repressor must first bind
to Trp hence tryptophan is a corepressor. In the absence of Trp the trp repressor
dissociates and transcription of the trp operon is initiated.

Attenuation

Attenuation regulates the termination of transcription as a function of tryptophan

concentration. At low levels of trp full length mRNA is made, at high levels
transcription of the trp operon is prematurely halted. Attenuation works by
coupling transcription to translation. Prokaryotic mRNA does not require
processing and since prokaryotes have no nucleus translation of mRNA can start
before transcription is complete. Consequently regulation of gene expression via
attenuation is unique to prokaryotes.

a. Attenuation is mediated by the formation of one of two possible stem-loop

structures in a 5' segment of the trp operon in the mRNA.

b. If tryptophan concentrations are low then translation of the leader peptide is

slow and transcription of the trp operon outpaces translation. This results in the
formation of a nonterminating stem-loop structure between regions 2 and 3 in the
5' segment of the mRNA. Transcription of the trp operon is then completed.

c. If tryptophan concentrations are high the ribosome quickly translates the mRNA
leader peptide. Because translation is occurring rapidly the ribosome covers region
2 so that it can not attach to region 3. Consequently the formation of a stem-loop
structure between regions 3 and 4 occurs and transcription is terminated.
 REGULATION IN PROKS VS EUKS

Because prokaryotic organisms lack a cell nucleus, the processes of

transcription and translation occur almost simultaneously. When the protein is
no longer needed, transcription stops. As a result, the primary method to control
what type and how much protein is expressed in a prokaryotic cell is through
the regulation of DNA transcription into RNA. All the subsequent steps happen
automatically. When more protein is required, more transcription occurs.
Therefore, in prokaryotic cells, the control of gene expression is almost entirely
at the transcriptional level.

Eukaryotic cells, in contrast, have intracellular organelles and are much more
complex. Recall that in eukaryotic cells, the DNA is contained inside the cell’s
nucleus and it is transcribed into mRNA there. The newly synthesized mRNA is
then transported out of the nucleus into the cytoplasm, where ribosomes
translate the mRNA into protein. The processes of transcription and translation
are physically separated by the nuclear membrane; transcription occurs only
within the nucleus, and translation only occurs outside the nucleus in the
cytoplasm. The regulation of gene expression can occur at all stages of the
process (Figure 2):

 Epigenetic level: regulates how tightly the DNA is wound around histone
proteins to package it into chromosomes
 Transcriptional level: regulates how much transcription takes place
 Post-transcriptional level: regulates aspects of RNA processing (such as
splicing) and transport out of the nucleus
 Translational level: regulates how much of the RNA is translated into protein
 Post-translational level: regulates how long the protein lasts after it has been
made and whether the protein is processed into an active form
 Table 1: Differences in the Regulation of Gene Expression of Prokaryotic and
Eukaryotic Organisms
Prokaryotic organisms
Lack nucleus
RNA transcription and
protein translation occur
almost simultaneously
Gene expression is regulated
primarily at the
transcriptional level
Eukaryotic organisms
Contain nucleus
RNA transcription occurs prior to protein
translation, and it takes place in the nucleus.
RNA translation to protein occurs in the
cytoplasm.
RNA post-processing includes addition of a 5′
cap, poly-A tail, and excision of introns and
splicing of exons.
Gene expression is regulated at many levels
(epigenetic, transcriptional, post-transcriptional,
translational, and posttranslational)