Last Update: 2 November 2017 Part I

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Control of Gene expression (lac - & trp- operon)
The total amount of DNA present in every cell may contain from a few to thousands of genes. The
different types of cells in the body of a multi cellular organism different in structure & function, their genes are
identical but expression time differ from one gene to other even with in the same cell thus the problem therefore
may arise:
How do cells with identical genetic complement different so much in structure and function?
The answer is that not all genes are active one time & all the protein is not needed at one time. Specific
proteins / enzymes are needed at different times in the life cycle of an organism therefore, it would be necessary
to have a mechanism which would allow only the desired genes to function at particular time. When necessary
the gene become activated while other remain inactive i.e. the genes are switch on and switch off at
different times & this process is called differential gene action.
Although a cell has genes to produce hundreds of enzymes, but only some enzymes are required at a
particular time which to be produced. This control mechanism ensures that the cell is not flooded with
unnecessary enzymes. This mechanism difficult to work out particularly in higher organisms, while in bacteria,
detail information has been obtained regarding regulation of gene expression. In eukaryotes only some
information has been made available in recent years.
A hypothesis to explain induction & repression
of enzyme synthesis was first put forward by F. Jacob
and J. Monod in 1961 for this and other some major
contribution in biochemistry they were awarded the
Nobel Prize in Medicine in 1965.
The Operon Model:
F. Jacob and J. Monod in 1961 on the basis of
their study on the inducible system for the synthesis of
galactosidase enzyme in E. coli, and proposed a model
in order to explain the induction or repression of
enzyme synthesis and this model is popularly known as Operon model.
Inducible system:
Figure 1 lac-operon model as proposed by Jacob &
Monod
The synthesis of -galactosidase, an enzyme
meant for hydrolysis of lactose in to glucose and
galactose, has been studied in considerable details.
galactosidase
Lactose glucose + galactose.

If the galactosides (lactose) are not supplied to

E. coli cells, the presence of -galactosidase is hardly
detectable, but as soon as lactose is added, the production
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of enzyme, -galactosidase increase as much as, 10,000 times. The enzyme quantity again falls down as quickly
as the lactose is removed.
Such enzymes, whose synthesis can be induced by adding the substrate, are known as inducible
enzymes and the genetic systems responsible for the synthesis of such an enzyme are known as inducible
system.
Repressible system:
In some other cases the situation is reverse to that of inducible system, for instance, when no amino acid
are supplied from out side, E. coli cells can syntheses all the enzyme needed for the synthesis of different amino
acid. However, if a particular amino acid like histidine is added, production of histidine synthesizing enzymes
will fall down. In such a system the addition of end product of a biosynthetic pathway will check synthesis of
the enzymes needed for the biosynthesis. Such enzymes, whose synthesis can be checked by adding end
product, are known as repressible enzymes and this genetic system would be known as repressible system.
So there are two systems,
1) Inducible system: Active repressor +
Inducers Inactive repressor.
2) Repressible system: Inactive repressor
+ Co-repressor Active repressor.
How they get such idea? They experiment
on E coli in respect their mode of nutrition
& enzymatic system.
a) Experiment 1: At first experiment
glucose introduced in to the medium &
found after short log-phase the glucose
enzyme production increase and it
detected by the essay as OD
observation.
b) Experiment - 2: When they lactose, a
disaccharide introduced into the
medium they found lag-phase is retarded then the glucose and thereafter OD increased.
c) Experiment 3: In the 3 rd experiment they collectively used glucose & lactose and found a diauxic curve
and indicate the first utilise glucose and after absence of any glucose then produced -galactosidase which
breakdown the disaccharides, lactose into glucose & galactose. From it they deducted hypothesis that there
must be a control mechanism by which the different enzyme synthesis takes place at different time. This is
nothing but an inducible enzyme they assumed that the synthesis of mRNA is associated with:
i. An operator gene which controlled the activity of numbers of structural genes
ii. Numbers of structural gene, which takes part the synthesis of protein.
The mechanism of regulation of protein synthesis utilizing operon model can be illustrated using the
example of two different operons (lac & tryptophan) in bacteria.

The Lac- Operon in E. coli :

The lac-operon contains a regulator, a promoter, a operator, and 3 structural genes, lac z, lac y and lac A
coding for the enzyme -galactosidase, -galactoside permease, and -galactoside transacetylase respectively.

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Fig. Diagram of major elements controlling the lactose operon

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Function of the structural genes:-
a) Gene-z codes for the enzyme galactosidase, which is active as a tetramer and breaks lactose into glucose & galactose.
b) Gene-y codes for the galactoside permease, which is a membrane bound protein and help in transport of metabolites.
c) Gene-a, which codes for -galactoside transacctylase, which is transfar, an acctyle group from Acctyle co-A to -galactosides.

Fig. 2 Fig. 3

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Fig. 4 Fig. 5

Fig. 6 Fig. 7

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Fig. 8
Fig. 9

Fig. 10 -6- Fig. 11

Fig. 12 Fig. 13

Fig. 14 -7- Fig. 15

Mutation in structural gene
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Any mutation in z and y genes gives lac genotype, which can not utilized lactose, while z mutant abolish
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enzyme activity, y mutants can not take up lactose from medium. No defect is observed in a mutants, which is a
puzzle.
Regulator gene
In lac-operon of E. coli repressor is synthesized through activity of gene i, known as regulator gene. The
repressor then attach on operator gene and check synthesis of mRNA. If lactose is added, the repressor is rendered
inactive so, that it cannot attach on operator gene and the synthesis of mRNA takes place in a non stop way.
Promoter gene
The activity of the regulator gene is controlled by a promoter gene, which is situated on its left side. It has
been shown that mutation in the promoter gene may permanently stop the mRNA synthesis of structural gene
product due to inability to bind the DNA polymerase enzyme with the promoter region.

Types of lac I Gene Mutations

A. Regulator Alleles :
1. Lac I mutants lac I+ gene is transdominan to lac I- gene (which is constitutive in its expression, i.e.
cannot bind to operator due to certain mutation on its operator binding site), for the partial diploid lacI+
lacO+ lac Z- lac Y+ / lac I- lac O+ lac Z+ lac Y- in absence of inducer the defective lac I- repressor is unable
to bind to either normal operator (lac O+) in the cell. But sufficient normal repressors, produced from the
lac I+ gene, are diffusing through the cell; they will bind to the two operators and hence block transcription
of with operons. In presence of inducer the wild type repressors become inactivated, so both operons are
transcribed.
i+ = makes a diffusible repressor protein that inhibits synthetic activity in any o + operon in the
absence of lactose; in the presence of lactose, repressor is inactivated
i- = a defective regulator that is unable to produce active repressor due to a nonsense or miss-sense
mutation
s
2. Lac I (Super repressor) mutants :- Shows no production of lac enzymes in presence or absence of
lactose (induces). In partial diploids with a lac I+ / lac Is, the lac Is allele is trans-dominant, affecting both
chromosomes. The super repressor protein is not in its ability to bind to the operator region but, instead,
cannot recognize the inducer allolactose therefore, the mutant super repressors get stuck on the operators,
and transcription of the operons can never occur. The presence of normal repressors in the cell has no effect
on this situation, since once a lac Is repressor is on the operator, nothing can remove it. As a consequence,
cells with a lac Is mutation cannot use lactose as a carbon source.
Is = makes a 'super repressor' , that is insensitive to lactose and inactivates any o+ operon

3. Lac I-d (dominance) mutants : Unlike the lac I- mutations, the lac I-d mutations are trans-dominant to lac
I+ in lac I-d / lac I+ partial diploids, so lac enzymes are produced constitutively even in the presence of lac
I+ gene. These mutations are clustered towards the 5 end of the lac gene. In haploid cells the lac I-d
mutants; the lac enzymes are made in the presence or absence of lactose.
4. The dominance of lac I-d mutants relates to the structure of the lac repressor. The repressor protein has four
identical polypeptides. There are relatively few, perhaps a dozen, repressor molecules in the cell. In the lac
I-d mutants, the repressor subunits do not combine normally, so no complete repressor is formed and no
operator specific binding is possible. The lac I-d / lac I+ diploids have a mixture of normal and mutant
polypeptides, which combine randomly to form repressor tetramers. The presence of one or more defective
polypeptides in the repressor is enough to block normal binding to the operator, So, there is a good chance
that no normal repressor proteins will be produced, since there are so few molecules per cell. As a result of
the absence (or virtual absence) of complete, functional repressors, a constitutive enzyme phenotype
results.

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 5. lac IQ and lac I SQ mutation :- Some mutations in the repressor gene promoter affect the expression of the
repressor gene itself. Since relatively few repressor molecules are synthesized in the wild type E. coli
cells, the repressor gene promoter must be of low affinity for RNA polymerase molecules. As in any other
region of the DNA, the repressor gene promoter is subject to base pair changes by mutation. Base pair
mutations have been found that decrease and the increase transcription rates.
The most useful mutants in this regard are lac IQ and lac ISQ mutants (where Q stands for quantity and
SQ for super quantity). Both mutations result in an increase in the rate of transcription of the repressor
gene, with the lac ISQ mutants giving the greater increase. Since lac IQ and lac ISQ mutants produce more
repressor molecules than the wild type, these mutants reduce the efficiency of induction of the lac-operon.
They can be induced, however, at very high lactose concentration.

B. Promoter Alleles
1. p+ = wild-type promoter; normal affinity for RNA polymerase
2. p- mutant promoter cannot bind RNA polymerase; none of the structural genes in the lactose operon are
transcribed
3. pS = increased affinity for recognition by RNA polymerase; elevates the transcriptional level of the operon
4. pi cr = affects the CRP-cAMP binding site to reduce the level of expression of lactose operon genes below
10% of wild type; i cr = insensitive to catabolite repression
C. Operator Alleles
1. O+ = in the absence of repressor, this operator' 'turns on' , the structural genes in its own operon; i.e., the z+
and y+ alleles in the same segment of DNA (cis position) can produce proteins; this operator is sensitive to
the repressor; i.e. , repressor will "turn off' , the synthetic activity of the structural genes in the lactose
operon
2. OC = a constitutive operator that is insensitive to repressor and permanently "turns on" the structural genes
in the lactose operon
D. Galactosidase Alleles
1. z+ = makes -galactosidase if its operon is "turned on" or "open"
2. z- = a missense mutation that makes a modified, enzymatically inactive product called Cz protein
3. z-ns = results in the destruction of the polycistronic message downstream from the mutation so that there is
no expression of any of the downstream lactose operon genes (a polar mutation); ns = nonsense
E. Permease Alleles
1. y+ = makes -galactoside permease if its operon is "turned on"
2. y- = no detectable permease is formed regardless of the state of the operator; probably a nonsense mutation

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