The Operon concept

The operon concept

• The basic concept for the way transcription is controlled in
bacteria
• Proposed by Francois Jacob and Jacques Monod in 1961
• Distinguished between two types of sequences in DNA
• Sequences that code for trans- acting products (usually
proteins)
• Cis-acting DNA sequences
• Gene activity is regulated by the specific interactions of the
trans-acting products with the cis-acting sequences
 Gene
• A sequence of DNA that codes for a diffusible product
• Either RNA or protein
• The product diffuses away from its site of synthesis to act
elsewhere
• Any gene product that is free to diffuse to find its target is
described as trans-acting
• Cis-acting applies to any sequence of DNA that functions
exclusively as a DNA sequence
• Affecting only the DNA to which it is physically linked
Components of genes and regulatory circuits
• Structural gene and regulator gene
• A structural gene is simply any gene that codes for a protein
(or RNA) product
• Protein structural genes represent an enormous variety of
structures and functions, including structural proteins,
enzymes with catalytic activities and regulatory proteins
• A type of structural gene is a regulator gene
• Which simply describes a gene that codes for a protein or an
RNA involved in regulating the expression of other genes
Components of genes and regulatory circuits
• A regulator gene codes for a protein that controls
transcription by binding to particular site(s) on DNA
• This interaction can regulate a target gene in:
• Positive manner (the interaction turns the gene on)
• Negative manner (the interaction turns the gene off)
• The sites on DNA are usually located just upstream (but not
exclusively) of the target gene
 Transcription Unit
• The sequences that mark the beginning and end of the
transcription unit- promoter and terminator (cis-acting sites)
• A promoter serves to initiate transcription only of the
gene(s) physically collected to it on the same stretch of DNA
• In the same way, a terminator can terminate transcription
only by an RNA polymerase that has traversed the preceding
gene(s)
• In their simplest forms, promoters and terminators are cis-
acting elements that are recognized by the same trans-
acting species (by RNAP)
 Negative control
• Transcription control in bacteria
• A repressor protein prevents a gene from being expressed
• Absence of the negative regulator, the gene is expressed
• Operator (cis-acting site) close to the promoter, binding site
for the repressor protein
• When the repressor binds to the operator
• RNA polymerase is prevented from initiating transcription
• Gene expression is turned off
 Negative control

• Trans-acting repressor binds to the

cis-acting operator
• Turn off transcription
 Positive control
• An alternative mode of control is positive control
• In bacteria (probably) equal frequency to negative control
• Most common mode of control in eukaryotes
• A transcription factor is required to assist RNAP in initiation
at the promoter
• Absence of the positive regulator, the gene is inactive
• RNAP cannot by itself initiate transcription at the promoter
 Positive control

• Trans-acting factor must

bind to the cis-acting site
• In order for RNAP to
initiate transcription at
the promoter
 Inducible gene
• A gene encodes an enzyme may be regulated by the conc. of
its substrate or product (or a chemical derivative of either)
• Bacteria need to respond swiftly to changes in their
environment
• Fluctuations in the supply of nutrients (sugars glucose or
lactose) can occur at any time
• Survival depends on the ability to switch from metabolizing
one substrate to another
• Economies are vital
• A bacterium indulges in energetically expensive ways to
meet the demands of the environment is at disadvantage
 Inducible gene
• Bacterium avoids synthesizing the enzymes of a pathway in
the absence of the substrate
• But, ready to produce the enzymes, if the substrate appear
• The synthesis of enzymes in response to the appearance of a
specific substrate
• Induction
• The gene is an inducible gene
 Repression
• The opposite of induction is repression
• Where the repressible gene is controlled by the amount of
the product made by the enzyme
• In E. coli, synthesizes the AA tryptophan by an enzyme
complex containing tryptophan synthetase and four enzymes
• If, however, tryptophan is provided in the medium on which
the bacteria are growing
• the production of the enzyme is immediately halted
• This allows the bacterium to avoid devoting its resources to
unnecessary synthetic activities
 Induction and repression
• Represent similar phenomena
• In one case the bacterium adjusts its ability to use a given
substrate (such as lactose) for growth
• In the other it adjusts its ability to synthesize a particular
metabolic intermediate (such as an essential amino acid)
• The trigger for either type of adjustment is a small molecule
that is:
• the substrate (or related to the substrate) for the enzyme
• or the product of the enzyme activity
 Inducer and corepressors
• Small molecules that cause the production of enzymes
• That are able to metabolize them (or their analogues)
• Inducers
• Those that prevent the production of enzymes
• That are able to synthesize them
• Corepressors
 Two ways of looking at regulation
• Negative versus positive control
• inducible versus repressible control
• Are combined to give four different patterns of gene
regulation
• Negative inducible
• Negative repressible
• Positive inducible
• Positive repressible
 Structural Gene Clusters are Coordinately
Controlled
• Bacterial genes are organized into operons
• Include genes that code for proteins whose functions are
related
• Genes coding for the enzymes of a metabolic pathway are
commonly organized into a cluster
 lac operon
• Cluster of the lac operon contains 3 lac structural genes
• lacZ, lacY and lacA
• Structural gene cluster is transcribed into a single
polycistronic mRNA from a promoter
• Where initiation of transcription is regulated
• The lac operon occupies -6000 bp of DNA
 Long lacZ gene starts at base 39
At the left the lac I gene has: lacY
Promoter lacA
Terminator terminator

End of the lacI region is Operator, O, occupies first 26 bp of the

adjacent to lacZYA promoter, P transcription unit
The protein products enable cells to take up and
metabolize β -galactoside sugars, such as lactose
 The roles of the three structural genes
• lacZ codes for the enzyme β -galactosidase
• Active form is a tetramer of -500 kD
• The enzyme breaks the complex β -galactoside into its
component sugars: Lactose
• Into glucose and galactose
• This enzyme also produces an important by product, β -1,6-
allolactase
 The roles of the three structural genes
• lacY codes for the β -galactoside permease
• 30-kD membrane-bound protein constituent of the transport
system
• Transports β -galactosides into the cell
• lacA codes for β -galactoside transacetylase
• Enzyme that transfers an acetyl group
• From acetyl-CoA to β-galactosides
 The roles of the three structural genes
• Mutations in either lacZ or lacY can create the lac genotype,
in which cells cannot utilize lactose
• The lacZ mutations abolish enzyme activity
• Directly preventing metabolism of lactose
• The lacY mutants cannot take up lactose efficiently from the
medium
 The roles of the three structural genes
• The entire system
• including structural genes
• the elements that control their expression
• forms a common unit of regulation
• an operon
• The activity of the operon is controlled by regulator gene(s)
whose protein products interact with the cis-acting control
elements
 The lac Operon: Negative Inducible
• Transcription of the lacZYA genes is controlled by a regulator
protein encoded by the LacI gene
• lacI comprises an independent transcription unit with its
own promoter and terminator
• lacI need not be located near the structural genes because it
specifies a diffusible product
• The lacI gene can function equally well if moved elsewhere,
or can be carried on a separate DNA molecule
• A trans-acting regulator
 The lac Operon: Negative Inducible
• The lacZYA genes are negatively regulated
• Transcribed, unless turned off by the regulator protein
• Repression is not an absolute phenomenon
• Turning off a gene is not like turning off a lightbulb
• Repression can be reduction in transcription by 5 or 100-fold
• A mutation that inactivates the regulator causes the structural
genes to be continually expressed, constitutive expression
• Product of lacI- lac repressor
• Its function is to prevent the expression of the lacZYA structural
genes
 The lac Operon: Negative Inducible
• Repressor is a tetramer of identical SUs of 38 kD each
• A wild-type cell contains ~ 10 tetramers
• Repressor gene is not controlled; it is an unregulated gene
• Transcribed into a monocistronic mRNA
• At a rate that appears to be governed simply by the affinity
of its (poor) promoter for RNA polymerase
 The lac Operon: Negative Inducible
• lacI is transcribed into a poor mRNA
• This is a common way to restrict the amount of protein
made
• In this case, the mRNA has no 5 ' UTR
• Which restricts the ability of a ribosome to start translation
• These two features account for the low abundance of lac
repressor protein in the cell
 The lac Operon: Negative Inducible
• Repressor functions by binding to an operator (Olac) at the
start of the lacZYA cluster
• Sequence of the operator includes an inverted repeat
• The operator lies b/w the promoter (Plac) and the structural
genes (lacZYA)
• When the repressor binds at the operator
• Prevents RNAP polymerase from initiating transcription at
the promoter
 The lac Operon: Negative Inducible
• Operator extends from
position -5 to +21
upstream of the mRNA
start point within the
transcription unit
• Overlaps the 3', right end
of the promoter
•A mutation that
inactivates the operator
• Causes constitutive
expression
 The lac Operon: Negative Inducible
• E. coli are grown in the absence of β-galactoside- no need for
β -galactosidase
• Contain very few molecules of the enzyme, about 5/cell
• Suitable substrate added, the enzyme activity appears very
rapidly in the bacteria
• Within 2-3 minutes, bacterium accumulates ~5000
molecules of enzyme
• Under suitable conditions, β -galactosidase can account for
5-10% of the total soluble protein of the bacterium
• Substrate removed from medium, the synthesis of the
enzyme stops as rapidly as it started
 Essential features of this induction
• Control of transcription of the lac operon responds very
rapidly to the inducer
• In the absence of inducer, the operon is transcribed at a very
low basal level
• Transcription is stimulated as soon as inducer is added
• The amount of lac mRNA increases rapidly to an induced level
• Reflects a balance between synthesis and degradation of the
mRNA
 Addition of inducer results in
rapid induction of lac mRNA
Removal of inducer,
rapid cessation of
A short lag by synthesis
synthesis of
the enzymes
Lac Repressor Controlled by a Small-Molecule
Inducer
• The ability to act as inducer or corepressor is highly specific
• Only the substrate/product of the regulated enzymes or
• a closely related molecule can serve this function
• For lac system the natural inducer is not lactose
• A byproduct of the LacZ enzyme, allolactose
• Also a substrate of LacZ enzyme, does not persist in the cell
• Inducers resemble the natural inducers of the lac operon but
cannot be metabolized by the enzyme
• Isopropylthiogalactoside (IPTG), an inducer
• not metabolized by β-galactosidase
 Lac Repressor Controlled by a Small-
Molecule Inducer
• Gratuitous inducers: Molecules that induce enzyme
synthesis but are not metabolized
• Component that represses the lac operon is the lac repressor
protein, encoded by lacI gene
• The lac repressor protein is induced by allolactose and IPTG
to allow expression of lacZYA
• The LacZ enzyme (β -galactosidase) utilizes allolactose and
lactose as substrates
• lacI is not induced by lactose
• LacZ enzyme does not metabolize IPTG
Lac Repressor Controlled by a Small-Molecule
Inducer
• The component that responds to the inducer is the
repressor protein encoded by lacI
• Its target, the lacZYA structural genes,
• Transcribed into a single mRNA from the promoter just
upstream of lacZ.
• State of the repressor determines whether this promoter is
turned off or on
Lac Repressor Controlled by a Small-Molecule
Inducer
• In the absence of an inducer, the
genes are not transcribed
• In an active form, repressor
protein is bound to the operator
• lac repressor maintains the lac
operon in the inactive condition
by binding to the operator
 Lac Repressor Controlled by a Small-Molecule
Inducer
• Inducer added, repressor is
converted into a form that leaves the
operator
• Addition of inducer converts
repressor to a form with low affinity
for the operator
• Allows RNAP to initiate transcription
• Transcription starts at the promoter
and proceeds through the genes to a
terminator located beyond the 3' end
of lacA
Lac Repressor Controlled by a Small-Molecule
Inducer
• Crucial features reside in the dual properties of the repressor:
• Can prevent transcription
• Can recognize the small-molecule inducer
• The repressor has two types of binding site:
• One type for the operator DNA
• One type for the inducer
Lac Repressor Controlled by a Small-Molecule
Inducer
• When the inducer binds at its site
• It changes the structure of the protein to influence the
activity of the operator-binding site
• The ability of one site in the protein to control the activity
of another
• Allosteric control