INSUCTION AND REPRESSION IN PROKARYOTES

Certain gene-product, such as tRNA molecules, rRNA molecules, ribosomal proteins, RNA
polymerase components (polypeptides), and other enzymes catalysing metabolic processes that are
frequently referred to as cellular “housekeeping” functions, are essential components of almost all
living cells. Genes that specify product of this type are continually being expressed in most cells.
Such genes are said to be expressed constitutively and are frequently referred to as constitutive
genes.
Other gene-products are needed for cell growth only under certain environment conditions.
Constitutive synthesis of such gene-products would clearly be wasteful, using energy that could
otherwise be utilized for more rapid growth and reproduction under the exiting environmental
conditions. The evolution of regulatory mechanisms that would provide for the synthesis of such
gene-product only when and where they were needed would clearly provide organism possessing
these regulatory mechanisms with a selective, advantage over organism lacking these mechanism.
This undoubtedly explains why presently existing organism, including the “primitive” bacteria and
viruses, exhibit highly developed and very efficient mechanism for the control of gene expression.
Escherichia coli and most other bacteria are capable of growth using any one of several
carbohydrates (e.g glucose, sucrose, galactose, arabinose, lactose) as an energy source. If glucose
is present in the environment, it will be preferentially metabolized by E. coli cells. In the absence
of glucose, however, E. coli cells can grow very on other carbohydrate. Cells growing in medium
containing the sugar lactose, for example, as the sole carbon source synthesize two enzymes, β-
galactosidase and β-galactoside permease, that are uniquely required for the catabolism of lactose.
(a third enzyme, β-galactoside transacetylase, is also synthesized. It has no known metabolic
function, however.) β-galactosidase cleave lactose into glucose and galactose, and β-galactoside
permease pump β-galactoside into the cell. Neither of these enzyms is of any use to E. cly cells
when present in environment not containing lactose. The synthesis of these two enzymes, of
course, requires the utilization of considerable energy (in the from ATP and GTP; see chapter 10).
Thus, E. coli cells have evolved a regulatory mechanism by which the synthesis of these lactose-
catabolizing enzymes is turned off in its absence.
In natural environments (intestinal tracts and sewers), E. coli cells probably encounter and absence
of glucose and the presence of lactose relatively infrequently. Most of the time , therefore, the E.
coli genes coding for enzymes involved in lactose are transferred to medium containing lactose as
the only carbon source, they rapidly begin synthesizing the enzymes required for lactose utilization
(Fig. 14.1a). This process, by which the expression of genes is turned on in response to a substance
in the environment, is called induction. Genes whose expression are so regulated are called
inducible genes, their product, if enzymes, are called inducible enzymes. The substances or
molecules responsible for induction are known inducers.
Enzyme that are involved in catabolic (degradative) pathways, such as in lactose, galactose or
arabinose utilization, are characteristically inducible. As will become apparent in the following
sections of this chapter, induction occurs at the level of transcription. It alters the rate of synthesis
of enzymes not the activity of existing enzymes molecules. Induction should nt be confused with
enzyme activation, in which the binding of a small molecule to an enzyme increases the activity
of the enzyme (but does not affect its rate of synthesis).
Bacteria possess the metabolic capacity to synthesize most of the organic molecules (such as amino
acids, purines, and vitamins) required for their growth. For example, E. coli has five coding for
enzyme that the required in the synthesis of tryptophan. These five genes must be expressed in E.
coli ells growing in an environment devoid of tryptophan in order to provide adequate amounts of
this amino acid for on-going protein synthesis.
When E. coli cells are present in an environment containing concentrations of tryptophan sufficient
to support optimal growth, the continued synthesis of the tryptophan biosynthetic enzyme would
be a waste of energy, because these bacteria have the capacity to take in external tryptophan. Thus,
a regulatory mechanism has evolved in E. col by which the synthesis of the tryptophan biosynthetic
enzymes is turned off when tryptophan is present the external milieu (Fig. 14.1b). This process of
“turning off” the expression of sets of genes is called repression. A gene whose expression has
been turned off this way is said to be repressed; when its expression is turned on, a gene of this
type is said to be derepressed.
Enzyme that are component of anabolic (biosynthetic) pathways are frequently subject to
repression (are repressible). Repression, like induction, occurs at level of transcription. Repression
should not be confused with feedback inhibition, in which the binding of an and product to the
first enzyme in a biosynthetic pathway inhibits the activity of the enzyme (but does not affect its
synthesis).
The Operon Model
Induction and repression of gene expression can be accomplished by essentially the same
mechanism. This mechanism was first accurately described in 1961 when F. Jacob and J. Monod,
both 1965 Nobel Prize recipients, proposed the operon model to explain the regulation of genes
encoding the enzymes required for lactose utilization in E. coli. Jacob and Monod proposed that
the transcription of one or a set of contiguous structural genes (genes coding for polypeptides) is
regulated by two controlling elements (Fig. 14.2a). One of these element, called the regulator gene
(or repressor gene), codes for a protein called the repressor, under the appropriate conditions, the
repressor bind to the second element, the operator (or operator sequence). The operator is always
located contiguous to the structural gene or genes whose expression it regulates. When the
repressor is bound to the operator, transcription of the structural genes cannot occur. We now know
that this result because the binding of the repressor to the operator sterically prevent RNA
polymerase from binding at the promoter site (the RNA polymerase binding site; see Chapter 10),
which is always located contiguous with (or even overlapping) the operator sequence. The operator
is usually located between the promoter and the structural genes (Fig. 14.2a). (The promoter was
not recognized at the time of Jacob and Monod’s proposal, but has since been shown to be an
essential component of an operon.) The complete contiguous unit, including the structural gene or
genes, the operator, and the promoter, is called an operon.
Whether the repressor will bind to the operator and turn off the transcription of the structural genes
in an operon is determined by the presence or absence of effector molecules (small molecules such
as amino acids and sugars) in the environment. In the case of inducible operons, these effector
molecules are called inducers. Those active on repressible operons are called co-repressors. These
effector molecules (inducers and co-repressors) act by binding to (or forming a complex with) the
repressors.
The only essential different between inducible operons and repressible operons is whether the
naked repressor or the repressor-effector molecule complex is active in binding to the operator. (1)
In the case of an inducible operon, the free repressor binds to the operator, turning off transcription
(Fig. 14.2b). When the effector molecule (the inducer) is present, it binds to the repressor, releasing
the repressor from the operator, that is, the repressor-inducer complex cannot bind to the operator.
Thus, the addition of inducer turn on or induces the transcription of the structural genes in the
operon (Fig. 14.2b). (2) In the case of a repressible operon, the situation is just reserved. The free
repressor-effector molecule (co-repressor) complex is active in binding to the operator (Fig. 14.2c).
Thus, transcription of the structural genes in a repressible operon is turned on in the absence of
and turned off in the presence of the effector molecule (co-repressor). Except for this difference in
the operator-binding behaviour of the repressor, inducible and repressible operons are comparable.
A single mRNA transcript carries the coding information of an entire operon. Thus, the mRNA of
operons consisting of more than one structural gene are polygenic. For example, the tryptophan
operon mRNA of E. coli is huge macromolecule carrying the coding sequence that specify five
different polypetides (see Fig 14.5). Because of their cotranscription, all the structural genes in an
operon are coordinately expressed.
Because the product of the regulator gene, the repressor, act by shutting off the transcription of
structural genes, the operon model, as originally proposed by Jacob and Monod, is referred to as a
negative control system, the product of regulator genes are required to turn on transcription. We
will discuss example of positive control mechanism later in this chapter.
Lac, an Inducible Operon
Jacob and Monod proposed the operon model largely as a result of their studies of the lac operon
of E. coli. More is known about the lac operon than any other operon. The lac operon contains a
promoter, an operator, and three structural genes z, y, and a, coding for the enzymes β-
galactosidase, β-galactoside permease, and β-galactoside transacetylase, reprectively (Fig. 14.3).
β-galactoside permease”pumps” lactose into the cell. Where β-galactosidase cleaves it into glucose
and galactose (Fig 14.4). The function of the transacetylase is still not clear.
The lac regulator gene, designated the ἰ gene, codes for a repressor that is 360 amino acids length.
The active form of the lac repressor, however is a tetramer that contains four copies of the ἰ gene
product. In the absence of the inducer, the repressor bind to the lac operator sequence, preventing
RNA polymerase from binding to the promoter and transcribing the structural genes (see Fig.
14.2b). A few molecules of the z, y, and a gene-products are synthesized in the uninduced state,
providing a low background level of enzyme activity. However, this background activity is
essential for induction of the lac operon, because the inducer of the operon, allolactose, is derived
from lactose in a eaction that is catalysed by β-galactosidase (Fig. 14.4). Once formed, allolactose
binds to the operator in so doing, it induces transcription of z, y, and a structural genes (see Fig
14.2b).
The lac ἰ gene, operator, and promoter were all initially identified genetically by the isolation of
mutations within these genetic units that rendered them nonfictional. Mutations within the ἰ gene
and the operator frequently result in the constitutive synthesis of the lactose-utilizing enzyme.
These mutations are designated ἰ- and oc , respectively. The ἰ- and oc constitutive mutations can be
distinguished not only by map position, but also by their behaviour in F’ merozygotes (see chapter
8, p.220) in which they are located in cis and trans configurations relative to mutations in lac
structural genes.
Merozygotes (partial diploids) of genotype F’ ἰ+ o+ z+ y+ a+ / ἰ+ o+ z- y- a- or of genotype F’ ἰ+ o+ z-
y- a- / ἰ+ o+ z+ y+ a+ are inducible for the utilization of lactose as a carbon source, just like haploid
wild-type (ἰ+ o+ z+ y+ a+) cells. The wild-type alleles (z+ y+ and a+) of the three structural genes are
dominant to their mutant alleles (z- y- and a-). This is to be expected because the wild-type alleles
produce functional enzymes, whereas the mutant alleles produce no enzymes or defective
(inactive) enzymes. F’ merozygotes that have the genotype ἰ+ o+ z+ y+ a+ / ἰ- o- z+ y+ a+ are also
inducible for the synthesis of the three enzymes specified by the lac operon. Thus ἰ+ is dominant
to ἰ- as expected, since ἰ+ specifies an active protein (repressor molecules) and ἰ- yields an inactive
protein.
Merozygotes that have the genotype F’ ἰ+ o+ z+ y+ a+/ἰ- o- z- y- a- or the genotype F’ ἰ+ o+ z- y- a- / ἰ-
o+ z+ y+ a+ are inducible for β-galactosidase, β-galactosidase permease, and β-
galatositransacetylase, like wild-type cells. This indicates that the ἰ gene codes for a diffusible
product, since it affects the expression of structural genes located in either cis or trans-
configuration in relation to itself. On the other hand, the operator constitutive of mutations act only
in cis, that is oc mutations cause only the constitutive expression of structural genes located on the
same chromosome. Of course, this is to be expected if the operator is the repressor binding site; as
such, it does not code for any product, diffusible or otherwise. A merozygotes of genotype F’ ἰ+ oc
z- y- a-/ἰ+ o+ z+ y+ a+ is thus inducible for the three enzymes specified by the structural genes of the
three enzymes specified by the structural genes if the lac operon whereas a merozygotes of
genotype F’ ἰ+ oc z+ y+ a+/ἰ+ o+ z- y- a- synthesized these enzymes constitutively.
Some of the ἰ gene mutations, those designated ἰ-d, are dominant to wild-type allele (ἰ+). This
dominance apparently result from the inability of heteromultimers (recall that the lac repressor
functions as a tetramer), which contain both wild-type and mutant polypeptides, to bind to the
operator sequence. Other ἰ gene mutations, those designated ἰ-s (for “superrepressed”), cause the
lac operon to be un-inducible. ( Strains carrying these ἰ-s mutations can usually be inducted to some
degree by using extremely high concentrations of inducer. They are not induced at normal inducer
concentrations.) When studied in vitro, the mutant ἰ-s polypeptides form tetramers that bind to lac
operator DNA. They either do not bind inducer, however, or exhibit a very low affinity for inducer.
The ἰ-s mutations therefore modify the inducer binding site of the lac repressor.
Promoter mutations do not alter the inducibility of the lac operon, instead they modify the levels
of gene expression in the induced and un-induced state by changing the frequency if initiation of
lac operon transcription (i.e., the efficiency of RNA polymerase binding).
The lac promoter actually contains two functionally distinct components: (1) the RNA polymerase
binding site and (2) a binding site for another protein. Called catabolite activator protein
(abbreviated GAP), that functions such as the lac operon is not transcribed in the presence of
glucose at concentrations sufficient to support optimal growth. This second control circuit assures
the preferential utilization of glucose as an energy source when it is available.
trp, a Repressible Operon
The trp (tryptophan) operon of E. coli is probably the best-known repressible operon. The
organization of the five structural genes and the adjacent regulatory sequence of the trp operon
(Fig.14.5) has been analysed in detail by Charles Yanofsky and colleagues. The regulation of
transcription of the trp operon occurs as diagrammed in Fig.14.2. The trp operon repressor is the
product of gene trpR, which is not closely linked to the trp operon Fig.14.5).
In the absence of tryptophan (the co-repressor), RNA polymerase binds to the promoter region and
transcribes the structural genes of the operon. In the presence of tryptophan, the co-
repressor/repressor complex bind to the operator region and prevents the binding of RNA
polymerase to the promoter. The operator sequence of trp operon lies entirely within the prompter
region(Fig. 14.5).

The rate of transcription of the trp operon in the derepressed state (absence of tryptophan) is 70
times the rate that occurs in the repressed state (presence of tryptophan). In trpR mutants that
cannot make repressor, the rate of synthesis of the tryptophan biosynthetic enzymes (the products
of the structural genes of the trp operon is still reduced about 10-fold by the addition of tryptophan
to the medium. This reduction is due to a second level of regulation of trp operon expression called
attenuation. Attenuation tryptophan-mediated termination of transcription in the trpL (mRNA
leader) region of the operon (see pp. 403-406).