Molecular biological

1. GENE REGULATION IN
PROKARYOTES
Year II
Biomedical Sciences
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2. Gene regulation
 The DNA sequence within each cell contains
the information required to synthesize
thousands of different proteins and RNA
molecules.
 Typically, a cell expresses only a fraction of its
genes.
 Different cell types in multicellular organism
arise because each type expresses a different
set of genes.
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3. Gene regulation
 bacterial cell lives in direct contact with its
environment, which may change dramatically in
chemical composition or temperature from one
moment to the next.
 At certain times, a particular food source may be
present, while at other times that compound is
absent
 Some molecules are absorbed from the immediate
environment or synthesized within the cell (e.g.,
lactose and tryptophan).
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4.  Genes may be regulated positively or
negatively.
 In positive regulation, a gene is incapable of
expression unless it receives a positive signal
of some sort.
 In negative regulation, a gene is inherently
active but is prevented from expressing itself
unless certain inhibitory factors are removed.
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5. Cont...
 Cells possess mechanisms that allow them to
precisely regulate their genetic information,
expressing genes only when they are needed
 When a bacterial cell encounters a potential
food source it will manufacture the enzymes
necessary to metabolize that food
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6.  In addition to sugars like glucose and lactose,
E. coli cells also require amino acids
 One essential amino acid is tryptophan.
 When E. coli is swimming in tryptophan it will
absorb the amino acids from the media
 When tryptophan is not present in the media
then the cell must manufacture its’ own amino
acid
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7. Prokaryotic Gene Structure
Prokaryotic Gene Structure
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Open reading frame(ORF)

8. Open Reading Frame (ORF)
 Open reading frame is a sequence from the
start till the first stop codon
 Ribosomes translate triplets of nucleotides
into amino acids
 Start codon is usually AUG (which is encoded
into methionine)
 Stop codons are UAA, UAG, UGA
 It is important to determine from which
nucleotide to start
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9. Gene regulation
 Gene regulation occurs at the level of
transcription or production of mRNA
 Prokaryotic genes are often organized such that
genes encoding related functions are clustered
together.
 This grouping of functionally related genes is
referred to as an operon.
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10. levels of regulation of gene expression
 A prokaryote, a unicellular organism, is totally
controlled by environmental changes
 Since transcription and translation in
prokaryotes are coupled, regulation mainly acts
at transcription level
 Prokaryotes adjust gene expression in response
to environmental conditions
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11. The operon
 An operon is a single transcription unit that
encodes multiple enzymes necessary for a
biochemical pathway
 Operons are transcribed to give single mRNA
molecules, each consisting of several genes.
 An operon are controlled by the Repressor or
Activator or inducer or substrate.
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12. The Operons
 The operon model was developed by François
Jacob and Jacques Monod (1961)
 An operon is composed of :
structural genes, control elements such as
operator sequences and regulator genes
(whose products recognize control elements).
 The expression of these genes are rapid and
synchronized.
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13. Cont...
 The purposes of the regulation of gene
expression in prokaryotes are remarkably well
served by the use of operons :
 All genes of an operon are coordinateley
expressed
 There is energy saving as the same set of
regulatory sequences and proteins is used for all
structural genes of the operon.
 These genes are transcribed as a polycistronic
transcript.
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14. Modulators of transcription
 The promoter is the site where the RNA polymerase binds
to the DNA prior to beginning transcription
 The operator typically resides adjacent to or overlaps with
the promoter and serves as the binding site for a protein,
usually a repressor .
 Repressors: mediate negative gene regulation, may
impede access of RNA polymerase to the promoter ther by
actively block transcription.
 Repressor binding is modulated by specific effectors
 The regulatory gene encodes the repressor protein
 Structural genes encode the enzymes of a particular
metabolic pathway.(eg.the Z,Y and a genes of lac operon)
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15. Cont...
 Activators: mediate positive gene regulation bind to
specific regulatory DNA sequences (e.g.enhancers)
o enhance the RNA polymerase -promoter interaction and
actively stimulate transcription common in eukaryotes
o Induction occurs when enzymes for a certain pathway
are produced in response to a substrate
 Inducers are small molecules that either activate or
repress transcription depending on the needs of the cell
and the availability of substrate.
 Inducers can help speed up or slow down “on” or “off”
by binding to a repressor or activator
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16. 2/5/2024 16

17. The lactose operon (lac operon)
 E. coli can use either glucose, which is a monosaccharide
or lactose, which is a disaccharide.
 However, lactose needs to be hydrolysed (digested) first
So the bacterium prefers to use glucose when it can.
 The lac operon consists of the genes that encode enzymes
necessary to utilize lactose
 The E. coli bacteria only needs beta galactosidase if there
is lactose in the environment to digest
 The enzymes responsible are only synthesized when
lactose is available as the sole source of carbon
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18. Lac Operon genes
 Lac Operon contains 3 genes for lactose breakdown
 Structural genes involved in lactose metabolism are:
 Lac Z codes for β-galactosidase hydrolyses lactose
into glucose and galactose
 lac Y codes for β-galactoside permease that which
regulates the lactose permeability in the cell.
 lac A codes for β-galactoside transacetylase that
eliminate toxic thiogalacosides
 Lac I gene is a regulatory gene that codes for
the repressor protein
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19. Cont...
 The lac operon composed of :
 Regulatory sequences :
 The operator which binds the repressor protein,
 The promoter containing two binding sites, one for the
RNA polymerase, the other for CAP-cAMP complex
 -galactosidase start codon (AUG) is at position +39 to +41
 Negative control is mediated by proteins called repressors.
 Positive control is mediated by another class of regulatory,
allosteric proteins called activators that can bind to DNA and
stimulate the initiation of transcription.
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20. Structure of lac operon
The lac operon consists of a promoter, an operator, and three genes (lac Z, Y,
and A) that encode proteins required for the metabolism of lactose.
In addition, there is a binding site for the catabolite activator protein (CAP),
which affects RNA polymerase binding to the promoter.
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21. Functional state of the E. coli lac operon growing
on lactose
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22. Action of the repressor
 Initiation of transcription of the lac operon is
controlled by the lac repressor protein.
 The repressor binds to the operator, which is
adjacent to the promoter .
 This binding of the repressor prevents RNA
polymerase from binding to the promoter.
 The repressor binds DNA in the absence of lactose,
but does not in the presence of lactose
 Thus DNA binding is sensitive to the presence of
lactose
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23. When lactose is not present
The repressor protein binds the operator site and this prevents/blocks
the RNA polymerase from binding the promotor and thus preventing
transcription
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24. When lactose is present
Lactose is converted to Allolactose which then binds the repressor
and alters the repressor’s shape so it cannot bind to the operator
site and block RNA polymerase activity and thus the enzymes to
break down lactose are produced
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25. The presence of glucose prevents induction
 If bacteria are grown in the presence of both
glucose and lactose, the lac operon is not
induced.
 As glucose is used up, the lac operon is induced
and this allows lactose to be used as an energy
source
 The activator, catabolite activator protein (CAP),
is an allosteric protein which binds cAMP as an
effector.
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26.  cAMP acts in prokaryotic cells by binding to a
protein, the cAMP receptor protein (CRP).
 By itself, CRP is unable to bind to DNA.
However, the cAMP-CRP complex recognizes
and binds to a specific site in the lac control
region
 The level of cAMP in cells is reduced in the
presence of glucose so that no stimulation of
transcription from CAP responsive operons
takes place
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27. Cont...
 The concentration of glucose in the cell affect the
activity of an enzyme called adenylate cyclase
 At high glucose concentration, adenylate cyclase
activity is inhibited.
 The decrease of glucose concentration activates this
enzyme which transforms ATP into cAMP
 Increasing amount of cAMP leads to cAMP bind
catabolite activator protein (CAP) making CRP-cAMP
complex
 CRP-cAMP complex binds to binding site(promotor)
and induces expression of the lac operon
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28. A.For CAP to bind to DNA, it must bind to cAMP.
When glucose levels are low, cAMP is
abundant and binds to CAP. The CAP–cAMP
complex causes the DNA to bend around it. This
brings CAP into contact with RNA polymerase
making polymerase binding to the promoter
more efficient.
B.High glucose levels produce two effects:
cAMP is scarce so CAP is unable to activate the
promoter, and the transport of lactose is
blocked
(inducer exclusion).
A
B
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29. Positive control of
the lac operon with
CAP
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30. tryptophan (or trp ) operon
 E. coli uses several proteins encoded by a cluster of 5
genes to manufacture the amino acid tryptophan
 All 5 genes are transcribed together as a unit called
an operon, which produces a single long piece of
mRNA for all the genes(polycistronic mRNA)
 RNA polymerase binds to a promoter located at the
beginning of the first gene and proceeds down the
DNA transcribing the genes in sequence
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31. Cont...
 The trp operon is continuously expressed in the
absence of tryptophan and is not expressed in the
presence of tryptophan.
 The trp repressor is a helix-turn-helix protein
that binds to the operator site located adjacent
to the trp promoter
 In the absence of tryptophan, the trp repressor
does not bind to its operator, allowing
expression of the operon and production of the
enzymes necessary to make tryptophan.
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32. Tryptophan Absent, Operon Derepressed
The tryptophan repressor alone cannot bind to DNA.
The promoter is free to function, and RNA polymerase transcribes
the operon.
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33. Tryptophan Present, Operon Repressed
When tryptophan is present, it binds to the repressor, altering its conformation
so it now binds DNA.
The tryptophan–repressor complex binds tightly to the operator, preventing RNA
polymerase from initiating transcription.
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34. Fig. 16.6
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35. Cont...
 When tryptophan is present, it binds the Trp repressor
and induces a conformational change in that protein,
enabling it to bind the trp operator and prevent
transcription.
 When the tryptophan concentration is low, the Trp
repressor is free of its co-repressor and vacates its
operator , allowing the synthesis of trp mRNA to
commence from the adjacent promoter.
 The ligand that controls the activity of the trp
repressor acts not as an inducer but as a co-repressor.
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36. Quorum sensing
• Quorum sensing is a cell–cell communication
process used by bacteria to coordinate gene
expression in response to changes in
population density
• This type of communication was originally
described in the marine luminescent
bacterium Vibrio fischeri.
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37. • V. fischeri colonize the light organ of
Euprymna scolopes.
• Inside the light organ, the bacteria grow to
high cell density and use quorum sensing to
activate expression of the luciferase operon.
The host benefits from this colonization
because it uses the bacterial-produced light to
counterilluminate itself to avoid predators.
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38. end
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