The document discusses gene regulation in prokaryotes, focusing on the lac operon in E. coli. It describes how the lac operon is regulated by a repressor protein, lactose as an inducer, and CRP/cAMP as a positive regulator. In the absence of lactose, the repressor binds to the operator region and prevents transcription. In the presence of lactose, it binds to the inducer and detaches from the operator, allowing transcription. CRP/cAMP activate transcription when glucose is low. The lac operon thus demonstrates negative and positive, inducible and repressible regulation depending on environmental conditions.

1. Gene regulation in Prokaryotes
By
Dr. YALAVARTHI NAGARAJU, Ph.D. (Ag.)

2. The DNA of prokaryotes is organized into a circular chromosome, supercoiled within the nucleoid
region of the cell cytoplasm.
Prokaryotic genome

3. E. Coli K12- Genome

4. Draft genome of E. coli K12

5. Gene regulation
• Gene regulation refers to the “ability of cells to control their level of
gene expression”
• Structural genes are regulated so proteins are only produced at certain
times and in specific amount
• Constitutive genes are unregulated and have essentially constant levels
of expression
• Benefits of gene regulation
1. Conserves energy- proteins produced only when needed
2. Ensures genes are expressed in appropriate cell type and at correct
stage in development

8. Types of gene expression
1. Constitutive expression
Some genes are essential and necessary for life, and therefore
they are continuously expressed, such as those enzymes involved in
TCA. These genes are called housekeeping genes
2. Induction and repression
The expression levels of some genes fluctuate in response to the
external signals

9. History
• In1960’s
Jacob and Monard are working on E. coli mutants
At the same time Lwoff is working on Phage λ
• Two key observations made by Jacob and Monard
1. Expression of three genes at higher level in the absence of lactose or
similar inducer-constitutive mutation
2. In other case, E. coli mutants couldn’t able to produce three enzymes
in the presence of lactose- Non-inducible mutation
Based on these observations Jacob, Monard and Lwoff proposed an
unifying hypothesis i.e., Gene regulation

10. • The hypothetical regulatory elements
1. Repressors
controlling messenger RNA (mRNA)
2. Operators
Jacob and Monard model of lac operon

11. Recent lac operon model

12. Operon-definition
• Proteins that are needed for a specific function, or that are involved in the same
biochemical pathway, are encoded together in blocks called “operons”
• Genes coding for proteins that function in the same pathway may be located
adjacent to one another and controlled as a single unit that is transcribed into a
polyscistronic mRNA
• Operon composed of structural genes, promoter, operator, and other regulatory
sequences

13. Components of lac operon- Repressor
• Which is encoded by the lacI gene, a tetrameric protein, each subunit
weighs about 38 kDa
• The lacI gene can function equally well if moved elsewhere, or can be
carried on a separate DNA molecule
• Repressors are controlled by a small molecule- inducer (allolactose)
• Binding of Repressor inhibit the transcription – Negative control
• The lacZYA genes are negatively regulated: They are transcribed unless
turned off by the regulator protein. Note that repression is not an
absolute phenomenon; turning off a gene is not like turning off a
lightbulb.
• Repression can often be a reduction in transcription by five-fold or
1OO-fold.
• Repressor has two binding sites, one for the operator DNA and another
for the inducer. Binding of IPTG or allolactose at this site inactivates the
repressor by vastly decreasing its affinity for DNA
• Repressor is inactivated by an allosteric interaction in which binding of
inducer at its site changes the properties of the DNA binding site
(allosteric control)
• The true inducer is allolactose, not an actual substrate of β-galactosidase
• lacI (repressor) is not induced by lactose

15. Repressor (tetramer)

16. Promotor
• A promoter serves to initiate transcription only of the gene(s)
physically connected to it on the same stretch of DNA

17. Operator
• Close to the promoter is another cis-acting site
called the operator, which is the binding site for the
repressor protein. When the repressor binds to the
operator, RNA polymerase is prevented from
initiating transcription, and gene expression is
therefore turned off.
• The operator extends from position -5 just
upstream of the mRNA start point to position +21
within the transcription unit; thus it overlaps the 3',
right end of the promoter.
• The sequence of operator includes an inverted
repeat
• The operator comprises 35 base pairs, including 28
base pairs of symmetrical sequence; that is, it
includes a sequence that is identical in both
directions (shaded in the diagram).

18. • The operator site has palindromic sequence which can be recognized
by repressor protein
• The transcriptional starting point is located within the repressor
binding sequence

19. Structural genes
• The structural genes direct synthesis of cellular proteins through mRNA and determine the sequence
of amino acids in the proteins synthesized.
• In the presence of inducer, all the three proteins began to accumulate simultaneously but at different
rates

20. • In lac-operon there are 3 structural genes z, y, a which transcribe one long polycistronic mRNA
molecule. This controls the synthesis of 3 proteins
1. lacZ= β-galactosidase –
• Splits lactose into glucose & galactose, and synthesizes the allolactose (Inducer)
• This enzyme is tetramer, weighs around 500 kDa
2. lacY= galactoside permease -
• facilitates the entry of lactose/β-galactosides into cell,
• a 30 kDa membrane bound protein
3. lacA= thiogalactoside transacetylase –
• appears in small quantities upon lactose induction.
• Transfers an acetyl group from acetyl co-A to β-galactosides
• lac mRNA disintegrates within 3 minutes

22. Lactose transporter

23. Inducer- allolactose
• β-galactosidase has two functions,
1. Hydrolysis of lactose to glucose and galactose
2. Intramolecular isomerization of lactose to
allolactose
• To study the lac operon in laboratory, one often uses
a synthetic inducer such as isopropyl β-
thiogalactoside (IPTG)
• ITPG induces lac operon but not cleaved by β-
galactosidase. Hence, its concentration doesn’t
change during an experiment
• Molecules that induce enzyme synthesis but are not
metabolized are called gratuitous inducers.

24. • When the inducer binds at its site, it
changes the structure of the protein in
such a way as to influence the activity of
the operator-binding site.
• The ability of one site in the protein to
control the activity of another is called
allosteric control.

25. CAP (catabolite activator protein)/CRP
• Some promoters, though, do not allow RNA polymerase to initiate
transcription without assistance from an ancillary protein.
• Such proteins are positive regulators, because their presence is necessary to
switch on the transcription unit.
• One of the most widely acting activators is CRP. This protein is a positive
regulator whose presence is necessary to initiate transcription at dependent
promoters.
• CRP is active only when bound to cAMP, which behaves as a classic small-
molecule inducer for positive control
• cAMP is synthesized by the enzyme adenylate cyclase. The reaction uses ATP
as substrate and introduces an internal 3'-5' link via a phosphodiester bond
• CRP is a dimer of two identical subunits of 22.5 kD (210 amino acid
residues), which can be allosterically activated by a single molecule of cAMP.
• A CRP monomer contains a DNA-binding region and a transcription -
activating region.

26. • cAMP binding alters the structure of CRP to
change the DNA-binding domain from one
that binds all DNA weakly to strong,
sequence-specific DNA binding.
• A CRP dimer binds to a site of -22 bp at a
responsive promoter. The binding sites
include variations of the 5 -bp consensus
sequence
• Adenylate cyclase activity is repressed by
high glucose
• Thus, the level of cAMP is inversely related
to the level of glucose.
• Only with low levels of glucose is the enzyme
active and able to synthesize cAMP.
• In turn, cAMP binding is required for CRP to
bind DNA and activate transcription.
• Thus, transcription activation by CRP only
occurs when cellular glucose levels are low.

27. Types of regulation
• Negative regulation- a repressor protein binds to an operator to prevent a
gene from being expressed
• Positive regulation- a transcription factor is required to bind at the
promotor in order to enable RNA polymerase to initiate transcription
• Inducible regulation- the gene is regulated by the presence of its substrate
(the Inducer)
• Repressible regulation- the gene is regulated by the product of its enzyme
pathway (the corepressor)
• We can combine these in all four combinations
1. Negative inducible
2. Negative repressible
3. Positive inducible and
4. Positive repressible

29. Lac regulation- in the presence of glucose (negative regulation)
• The lac repressor (Laci) –operator system
keeps the operon turned off in the absence
of utilizable β-galactosides.
• E. coli has long been known to use glucose
in preference to most other energy
substrates.

30. Lac regulation - in the presence of Glucose and Lactose (positive regulation)
• When grown in a medium containing both
glucose and lactose, the cells metabolize
glucose exclusively until the supply is
exhausted.
• Then growth slows, and the lactose operon
becomes activated in preparation for continued
growth using lactose. This phenomenon, now
known to involve a transcriptional activation
mechanism, was originally called glucose
repression or catabolite repression.
• When glucose levels drop, cAMP levels rise.
cAMP interacts with a protein called cAMP
receptor protein (CRP), triggering activation of
the lactose operon.

31. cAMP-CAP binding to DNA

32. Lac regulation- in the presence of Lactose (inducible regulation)
• One tetramer of repressor usually is bound to the
operator.
• In a noninduced cell a tetramer is bound at the
operator, whereas the remaining repressor
molecules are bound to nonspecific sites.
• There are likely to be very few or no repressor
tetramers free within the cell.
• The effect of induction is therefore to change the
distribution of repressor on DNA, rather than to
generate free repressor.
• The addition of inducer abolishes the ability of
repressor to bind specifically at the operator.
• Those repressors bound at the operator are
released and bind to random (low-affinity) sites.
Thus, in an induced cell, the repressor tetramers
are "stored" on random DNA sites.

33. Inducer-Repressor Binding

34. Lac regulation -in the absence of Lactose and Glucose (repressible regulation)
• CRP is active and binds with the lac operon due to high availability of cAMP.
• Lac repressor remains bound to the operator and prevents transcription

35. Lac operon responses
Glucose Lactose Binding of CAP Binding of repressor Level of transcription
+ - - + No transcription
+ + - - Low level Transcription
- + + - High level transcription
- - + + No transcription

36. Overall picture of lac operon operation

37. Paradox in lac operon
• The lac operon contains the structural gene (lacZ) coding for the β-galactosidase
activity needed to metabolize the sugar; it also includes the gene (lacY) that codes
for the protein needed to transport the substrate into the cell. If the operon is in a
repressed state, how does the inducer enter the cell to start the process of induction?
• β-galactosidase (encoded by lacZ) is required to make the inducer allolactose to
induce the synthesis of β-galactosidase.
How is allolactose synthesized to allow induction of the gene? (An operon with a
mutant lacZ gene cannot be induced.)
• The answer is….
The operon has a basal level of expression, ensuring that a minimal amount of
LacZ and LacY proteins are present in the cell-enough to start the process. Even when
the lac operon is not induced, it is expressed at a residual level (0.1% of the induced
level). In addition, some inducer enters the cell via another uptake system. The basal
level of 13-galactosidase then converts some lactose to allolactose, leading to
induction of the Lac operon.