The document describes the lac operon in E. coli, which regulates genes involved in lactose metabolism. The lac operon contains three structural genes - lacZ, lacY, and lacA - that are regulated by the lac repressor and catabolite activator protein (CAP). The lac repressor normally binds to the operator region and blocks transcription. In the presence of lactose, allolactose binds to the lac repressor and causes it to detach from DNA. CAP promotes transcription when glucose levels are low, allowing the cell to preferentially metabolize lactose over glucose. Together, these regulatory mechanisms ensure the lac genes are only expressed when lactose is available and glucose is not.

1. Y4E01-BASIC MOLECULAR
BIOLOGY
Dr. s. sivasankara narayani
Assistant professor
Department of Microbiology
Ayya nadar Janaki ammal college
sivakasi
12-01-2021Dr.SS ., MRSB (UK)

2. UNIT - I
• Nucleic acid: DNA as the genetic material – direct and indirect
evidences. RNA as the genetic material. Structure, types and
properties of DNA and RNA. Chargaff’s rule. Genetic code:
Properties – codon - anticodon - *Wobble hypothesis. Law of DNA
constancy and C-value paradox.
12-01-2021Dr.SS ., MRSB (UK)

3. UNIT - II
• DNA replication: Theories - rolling circle mechanism –
semiconservative - conservative and discontinuous- evidences.
Enzymes involved in replication of DNA in prokaryotes and
eukaryotes.
12-01-2021Dr.SS ., MRSB (UK)

4. UNIT - III
• Transcription process in prokaryotes and eukaryotes: Initiation -
elongation - termination. RNA processing in eukaryotes: Capping -
polyadenylation - *splicing. Translation in prokaryotes and
eukaryotes: Initiation - elongation - termination. Post translational
modification in eukaryotes.
12-01-2021Dr.SS ., MRSB (UK)

5. UNIT - IV
Regulation of gene expression in bacterial system : Operon concept
– lac, trp, ara operon in E. coli - *DNA methylation and
heterochromatization. Transposons (brief account only).
12-01-2021Dr.SS ., MRSB (UK)

6. UNIT- V
Mutation : Types - molecular basis of spontaneous and induced
mutations. Mutagenic agents: Chemical and physical mutagens.
Isolation of auxotrophs and drug resistance mutant. Toxicity testing-
*Ames test. DNA damage and repair (photoreactivation, excision).
12-01-2021Dr.SS ., MRSB (UK)

7. LAC OPERON
• The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed
only when lactose is present and glucose is absent.
• Two regulators turn the operon "on" and "off" in response to lactose and glucose levels:
the lac repressor and catabolite activator protein (CAP).
• The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon,
but stops acting as a repressor when lactose is present. The lac repressor senses lactose
indirectly, through its isomer allolactose.
• Catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of
the operon, but only when glucose levels are low. CAP senses glucose indirectly, through
the "hunger signal" molecule cAMP.
12-01-2021Dr.SS ., MRSB (UK)

8. OPERON
• Operon is operating units which can be defined as the cluster of
genes located together on the chromosomes & transcribed
together.
• It is group of closely linked structure genes & associated control
gene which regulate the metabolic activity.
• All the genes of an operon are coordinately controlled by a
mechanism 1st described in 1961 by Francois Jacob & Jaques Monod
of the Pasture institute of Paris.
12-01-2021Dr.SS ., MRSB (UK)

9. LAC OPERON
• The lactose operon designated as lac operon.
• The lac operon codes for enzymes involved in the catabolism
(degradation) of lactose.
• lactose is the disaccharide which is made up of glucose &
galactose.
• It is the inducible operon since the presence of lactose induce the
operon to switched on.
12-01-2021Dr.SS ., MRSB (UK)

10. STRUCTURE
• The lac operon contains three genes: lacZ, lacY, and lacA. These genes are
transcribed as a single mRNA, under control of one promoter.
• Genes in the lac operon specify proteins that help the cell utilize lactose.
• lacZ encodes an enzyme that splits lactose into monosaccharides (single-unit sugars)
that can be fed into glycolysis.
• Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose
into the cell.
• In addition to the three genes, the lac operon also contains a number of regulatory
DNA sequences. These are regions of DNA to which particular regulatory proteins
can bind, controlling transcription of the operon.
12-01-2021Dr.SS ., MRSB (UK)

11. . Structural genes of lac operon:
• lac Z codes for the enzyme β-galactosidase, a tetramer of about 500 kD.
• This enzyme breaks down β-galactoside into its monosaccharide components.
For example, lactose is split into glucose and galactose which can be
metabolized further through glycolysis.
ii. lac Y codes for the β-galactoside permease, a 30 kD membrane-bound protein
which transports β-galactosides into the cell.
iii. lac A codes for β-galactoside transacetylase, that transfers an acetyl group
from acetyl-CoA to β-galactosides (role in lac operon not clear).
12-01-2021Dr.SS ., MRSB (UK)

12. Regulatory genes
• The regulatory genes of lac operon includes promoter gene, operator gene, lac I, and catabolite
activator protein (CAP) binding site.
i. The promoter is the binding site for RNA polymerase, the enzyme that performs transcription.
ii. The operator is a negative regulatory site where the lac repressor protein binds. It is located between
the promoter and the structural genes and overlaps with the promoter.
iii. Lac I (repressor) gene codes for the lac operon repressor, which is a tetramer of identical subunits
of 38 kD each. This gene is located adjacent to the promoter of the lac operon, with its own
promoter and terminator and is always transcribed, hence the repressor is always synthesized.
iv. The repressor is a diffusible product, making Lac I is a trans-acting gene. Repressor binds the operator
to repress (turn off) the operon.
v. Catabolite Activator Protein (CAP) binding site is a positive regulatory site located just upstream of
the lac operon promoter, where the catabolite activator protein (CAP) binds.
vi. The CAP is a dimer protein, which has binding sites for cAMP and DNA. When cAMP binds CAP,
its affinity for the DNA increases. When bound to DNA, CAP promotes transcription by aiding RNA
polymerase bind to the promoter more efficiently.
12-01-2021Dr.SS ., MRSB (UK)

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14. LAC OPERON
• The promoter is the binding site for RNA polymerase, the enzyme
that performs transcription.
• The operator is a negative regulatory site bound by
the lac repressor protein. The operator overlaps with the
promoter, and when the lac repressor is bound, RNA polymerase
cannot bind to the promoter and start transcription.
• The CAP binding site is a positive regulatory site that is bound by
catabolite activator protein (CAP). When CAP is bound to this site,
it promotes transcription by helping RNA polymerase bind to the
promoter.
12-01-2021Dr.SS ., MRSB (UK)

15. The lac repressor
• The lac repressor is a protein that represses (inhibits)
transcription of the lac operon. It does this by binding to the
operator, which partially overlaps with the promoter. When bound,
the lac repressor gets in RNA polymerase's way and keeps it from
transcribing the operon.
• When lactose is not available, the lac repressor binds tightly to
the operator, preventing transcription by RNA polymerase.
However, when lactose is present, the lac repressor loses its
ability to bind DNA. It floats off the operator, clearing the way for
RNA polymerase to transcribe the operon.
12-01-2021Dr.SS ., MRSB (UK)

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17. …
• This change in the lac repressor is caused by the small
molecule allolactose, an isomer (rearranged version) of lactose.
When lactose is available, some molecules will be converted to
allolactose inside the cell. Allolactose binds to the lac repressor
and makes it change shape so it can no longer bind DNA.
• Allolactose is an example of an inducer, a small molecule that
triggers expression of a gene or operon. The lac operon is
considered an inducible operon because it is usually turned off
(repressed), but can be turned on in the presence of the inducer
allolactose.
12-01-2021Dr.SS ., MRSB (UK)

18. Catabolite activator protein (CAP)
• When lactose is present, the lac repressor loses its DNA-binding ability. This
clears the way for RNA polymerase to bind to the promoter and transcribe
the lac operon. That sounds like the end of the story, right?
• Well...not quite. As it turns out, RNA polymerase alone does not bind very well
to the lac operon promoter. It might make a few transcripts, but it won't do
much more unless it gets extra help from catabolite activator protein (CAP).
CAP binds to a region of DNA just before the lac operon promoter and helps RNA
polymerase attach to the promoter, driving high levels of transcription.
12-01-2021Dr.SS ., MRSB (UK)

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20. • CAP isn't always active (able to bind DNA). Instead, it's regulated by a small
molecule called cyclic AMP (cAMP). cAMP is a "hunger signal" made by E.
coli when glucose levels are low. cAMP binds to CAP, changing its shape and
making it able to bind DNA and promote transcription. Without cAMP, CAP
cannot bind DNA and is inactive.
• CAP is only active when glucose levels are low (cAMP levels are high). Thus,
the lac operon can only be transcribed at high levels when glucose is absent.
This strategy ensures that bacteria only turn on the lac operon and start using
lactose after they have used up all of the preferred energy source (glucose).
12-01-2021Dr.SS ., MRSB (UK)

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25. Glucose Lactose CAP binds
Repressor
binds
Level of
transcription
+ - - + No transcription
+ + - -
Low-level
transcription
- - + + No transcription
- + + -
Strong
transcription
Summary of lac operon responses
12-01-2021Dr.SS ., MRSB (UK)

26. sivasan91@gmail.com
12-01-2021Dr.SS ., MRSB (UK)

27. QUESTIONS TO THINK
• Operon
• Structural genes of lac operon
• Functional genes of lac operon
• Mechanism behind presence lactose
• Mechanism behind presence of glucose
12-01-2021Dr.SS ., MRSB (UK)

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