MB 206 : Module 1 - B

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MB 206 Module 1 - B

• Regulation of Gene Expression in Bacteria

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This diagram is for eukaryote
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Regulation of Gene Expression

• A cell contains the entire genome of an organism
  ALL the DNA.
• Gene expression transcribing and translating
  the gene
• Regulation allows an organism to selectively
  transcribe (and then translate) only the genes it
  needs to.
• Genes expressed depend on
• the type of cell
• the particular needs of the cell at that time.

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Principles of Gene Regulation
In genetics, constitutive refers to a gene
product made all the time. In the absence of
the activator or the repressor, RNA polymerase
transcribes the gene constitutively
A gene is expressed in higher level under
influence of some signal
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How Are Genes Regulated?

• Genes located in coherent packages called operons
• operons has 4 parts
• regulatory gene - controls timing or rate of
  transcription
• promoter - starting point
• operator - controls access to the promoter by RNA
  polymerase
• structural genes
• NOTE operons regulated as units

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Gene Regulation in Prokaryotes

• Prokaryotes organize their genome into operons
• Operon a group of related genes
• One promoter sequence at the very beginning
• All of the genes will be transcribed together (in
  one long strand of RNA.

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Principle of Gene Regulation

• RNA polymerase binds to DNA at promoters.
• Transcription initiation is regulated by proteins
  that bind to or near promoters.
• Repression of a repressible gene (i.e., negative
  regulation) repressors (vs activitors) bind to
  operators of DNA.
• Repressor is regulated by an effector, usually a
  small molecules or a protein, that binds and
  causes a conformational change.
• Activitor binds to DNA sites called
• enhancer to enhance the RNA
• polymerase activity.
• (i.e., positive regulation)
• Induction of an inducible gene, e.g., heat-shock
  genes.

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General organization of an inducible gene
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Regulation of Genes

Transcription Factor (Protein)
RNA polymerase
DNA
Regulatory Element
Gene
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Regulation of Genes

New protein
RNA polymerase
Transcription Factor
DNA
Gene
Regulatory Element
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Gene Expression
How much protein is in a cell (and active)??
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Most genes are not expressed at a particular time

• Not all of the genes in a bacteria will be
  expressed at the same time.
• Even in some of the smallest bacteria, about 500
  different genes exists
• Of the 4279 genes in E. coli , only about 2600
  (60) are expressed in standard laboratory
  conditions.
• Only about 350 genes are expressed at more than
  100 copies (i.e. molecules!) per cell, making up
  90 of the total protein.

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Possible target in control of gene expression
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Topics

• Lac Operon (Negative control Catabolic
  repression)
• Tryptophan Operon (Positive control)
• Histidine Operon (Attenuator)

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Comparison of genomes of various organism

ORGANISM coding Size of genome (bp) number of genes
Escherichia coli 90 4,639,221 bp 4288
Mycoplasma genitalium 88 580,073 bp 468
Haemophilus influenzae 86 2,087,778 bp 1,662
Methanococcus jannaschii 85 1,660,000 bp 1,997
Synechocystis sp. (PCC 6803) 80 3,570,000 bp 3,168
Saccharomyces cerevisiae 75 13,000,000 bp 6,275
Humans 2 3,000,000,000 bp 70,000 (?)
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Diploid numbers of some commonly studied organisms(as well as a few extreme examples) Diploid numbers of some commonly studied organisms(as well as a few extreme examples)
Homo sapiens (human) 46
Mus musculus (house mouse) 40
Drosophila melanogaster (fruit fly) 8
Caenorhabditis elegans (microscopic roundworm) 12
Saccharomyces cerevisiae (budding yeast) 32
Arabidopsis thaliana (plant in the mustard family) 10
Xenopus laevis (South African clawed frog) 36
Canis familiaris (domestic dog) 78
Gallus gallus (chicken) 78
Zea mays (corn or maize) 20
Muntiacus reevesi (the Chinese muntjac, a deer) 23
Muntiacus muntjac (its Indian cousin) 6
Myrmecia pilosula (an ant) 2
Parascaris equorum var. univalens (parasitic roundworm) 2
Cambarus clarkii (a crayfish) 200
Equisetum arvense (field horsetail, a plant) 216
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Genes in E.coli
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E.coli genes expressed

• A total of 4288 genes in Escherichia coli
• - 2600 genes found under standard
  laboratory growth conditions
• - 2100 protein spots detected under 2-D
  protein gels
• - 350 proteins in high amount, the rest
  are very low amounts
• Majority of the genes are likely to be expressed
  transiently, in small amounts during DNA
  replication, and then remain silent (unexpressed)
  until the next round of DNA synthesis

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Why is there a need to control gene expression?

• 1) Prevent energy wastage
• 2) Ensure only necessary proteins are made
  according to the requirement for cells growth.
• Small portion of DNA in cell used for genetic
  message (mRNA), the rest for regulatory purposes.

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Gene Regulation in bacteria

• How do single-celled prokaryotes like E. coli
  know how to respond to their environments?
• Each environmental cue generates a specific
  response, with specific proteins and reactions.
• eg.
• A bacterium can use different sources of
  Nitrogen
• - incorporate N2 gas from the air
• - incorporate ammonia from their
  surroundings or
• - from amine group of an amino acid like
  glutamine (easier and less energy)
• These processes involve very different
  enzymes. Presence of glutamine, the cell will
  turn off synthesis of enzymes for fixing N2

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How can the cell "turn off" the synthesis of
proteins from its DNA?

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Gene regulation can occur at any place along the
flow of information from DNA to RNA to protein
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Different forms of gene regulation

• a. Regulation by DNA Replication (default)
• b. Transcriptional Regulation by different
  s-factors.
• c. Negative Regulation of Gene Expression
• d. Positive Control of Gene Regulation
• e. Alternative splicing of RNA (almost
  exclusively for eukaryotes)
• f. Post-transcriptional regulation
• - termination of transcription
• - translation control - message
  stability - protein stability

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E.coli RNA Polymerase subunits

Gene Mass KDa Use -35 Sequence separation -10 Sequence
rpoA 40 a subunit - - -
rpoB 155 b subnit - - -
rpoC 160 b' subunit - - -
rpoD 70 s70 General TTGACA 16-18 bp TATAAT
rpoN 54 s54 Nitrogen CTGGNA 6 bp TTGCA
rpoS 38 s38 Stationary not known not known not known
rpoH 32 s32 Heat shock CCCTTGAA 13-15 bp CCCGATNT
fliA 28 s28 Flagellar CTAAA 15 bp GCCGATAA
rpoE 24 s24 High temp. heat shock not known not known not known
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Transcription regulation by s-factors

• s70 - RpoD normal s-factor s54 - RpoN
  Nitrogen response s38 - RpoS Stationary
  phase s32 - RpoH Heat shock response s28
  - FliA Flagellar genes regulation s24 -
  RpoE Heat shock high temp.
• Approx 1500 - 2000 copies of RNAP holoenzyme/
  cell
• For bacteria growing in "log phase"
• 600 copies of RpoD (s70)
• 200 copies of RpoS (s38)
• RpoS increases to 600 copies per cell in
  stationary phase or osmotic shock.

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Operon and Regulon

• An operon
• - consists of a set of genes expressed
  coordinately transcribed
• as a single unit
• - Specific regulation (positive / negative)
  can induce or repress a
• particular gene or operon
• - contains both a regulatory a message
  region.
• - Regulatory / control region at the 5
  side of the gene codes for a
• protein (message region).
• Regulon
• - comprise of global regulation affecting a
  set of operons.
• - All operons in the regulon are
  coordinately controlled by the
• same regulatory mechanism.

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Operons-the basic concept of Prokaryotic Gene
Regulation

• Regulated genes can be switched on and off
  depending on the cells metabolic needs
• Operon a regulated cluster of adjacent
  structural genes, operator site, promotor site,
  and regulatory gene(s)

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Repressible vs. Inducible Operonstwo types of
negative gene regulation

• Repressible Operons
• Genes are initially ON
• Anabolic pathways
• End product switches off its own production

• Inducible Operons
• Genes are initially OFF
• Catabolic pathways
• Switched on by nutrient that the pathway uses

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lac an inducible operon
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The lac Operon of E. coli

• 1. Growth and division genes of bacteria are
  regulated genes. Their expression is controlled
  by the needs of the cell as it responds to its
  environment with the goal of increasing in mass
  and dividing.
• 2. Genes that generally are continuously
  expressed are constitutive genes (housekeeping
  genes). Examples include protein synthesis and
  glucose metabolism.
• 3. All genes are regulated at some level, so that
  as resources dwindle the cell can respond with a
  different molecular strategy.
• 4. Prokaryotic genes are often organized into
  operons that are cotranscribed. A regulatory
  protein binds an operator sequence in the DNA
  adjacent to the gene array, and controls
  production of the polycis-tronic (polygenic)
  mRNA.
• 5. Gene regulation in bacteria and phage is
  similar in many ways to the emerging information
  about gene regulation in eukaryotes, including
  humans. Much remains to be discovered even in E.
  coli, one of the most closely studied organisms
  on earth, 35 of the genomic ORFs have no
  attributed function.

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The lac Operon of E. coli

• Animation Regulation of Expression of the lac
  Operon Genes
• 1. An inducible operon responds to an inducer
  substance (e.g., lactose). An inducer is a small
  molecule that joins with a regulatory protein to
  control transcription of the operon.
• 2. The regulatory event typically occurs at a
  specific DNA sequence (controlling site) near the
  protein-coding sequence (Figure 16.1).
• 3. Control of lactose metabolism in E. coli is an
  example of an inducible operon.

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Lac Operon

• Transcription is OFF
• When there is no lactose that needs to be
  digested
• lacI repressor is in active form ? binds to
  operator ? blocks RNA Polymerase ? no
  transcription

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Lac Operon

• Transcription is ON
• When there is lactose that needs to be digested
• Lactose binds to lacI repressor ? inactivates it
• RNA Polymerase is able to bind to promoter ?
  transcribe genes

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Negative Regulation of Gene Expression

• By default, the gene is usually
• switched ON.
• Binding of a REPRESSOR will switch
• the gene OFF.
• Most common regulation in BACTERIA
• Often this is found as AUTOREGULATION - where too
  much of the gene product inhibits further
  transcription - usually this is through binding
  to the upstream promoter control region.
• A good "classic" example is the E.coli lac
  operon.

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Positive control of Regulation

• By default, the gene is usually
• switched OFF.
• Binding of a ACTIVATOR will switch
• the gene ON. (often transcriptional
  activators / factors
• bind and bend DNA upstream of the
  promoter.)
• Most common in EUKARYOTES
• Some promoters are not very functional in the
  absence of a transcriptional activator
  protein(s).

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Lac Operon

• Lactose metabolism occurs when the environment
  contains lactose.
• Enzymes required for lactose degradation are
  TURNED ON.
• beta-galactosidase (lac Z)
• - enzyme hydrolyzes the bond between glucose
  galactose.
• Lactose Permease (Lac Y)
• - enzyme spans the cell membrane
• - transports lactose into the cell from
  the outside environment.
• - Membrane is otherwise essentially
  impermeable to lactose.
• Thiogalactoside transacetylase (LacA)
• - The function of this enzyme is not known.
  

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Lactose metabolism
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Regulatory elements in the Lac Operon
Element
Function Operator (LacO) binding site for
repressor Promoter (LacP) binding site
for RNA polymerase Repressor (LacI)
codes for lac repressor protein
Binds to DNA at
operator and blocks binding of
RNA polymerase at promoter Pi promoter
for Lac I CAP
binding site for cAMP/CAP complex
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• Glucose or Lactose?
• A bacterium's prime source of food is glucose,
  since it does not
• have to be modified to enter the respiratory
  pathway.
• So if both glucose lactose are around, the
  bacterium will
• to turn off lactose metabolism in favor of
  glucose metabolism.
• There are sites upstream of the Lac genes that
  respond
• to different glucose concentrations.

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Presence of inducer - lactose
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Regulation of Lac operon - depending on
availability of lactose or glucose
Low levels of Glucose / Catabolite repression
Absence of lactose
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• Regulation of Lac Operon
• When lactose is present, it acts as an inducer of
  the operon. Lactose enters the cell and binds to
  the Lac repressor, inducing a conformational
  change preventing the repressor from binding to
  the operator. This allows the RNA polymerase
  binding at the promoter to proceed with
  transcription of mRNA (LacZ, LacY LacA) and
  production of enzymes for the metabolism of
  lactose.
• When the inducer (lactose) is removed, the
  repressor returns to its original conformation
  and binds to operator, blocking the RNA
  polymerase from proceeding with transcription of
  mRNA, thus no protein is made.
• The lac operon is always primed for transcription
  upon the addition of lactose.
• When levels of glucose (a catabolite) in the cell
  are high, formation of cyclic AMP is inhibited.
  But when glucose levels drop, more cAMP forms.
  cAMP binds to a protein called CAP (catabolite
  activator protein), which is then activated to
  bind to the CAP binding site. This activates
  transcription, by increasing the binding affinity
  of RNA polymerase to promoter. This is called
  catabolite repression, a misnomer since it
  involves activation, but understandable since it
  seemed that the presence of glucose repressed all
  the other sugar metabolism operons.

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The Tryptophan Operon (Positive regulation)

• Trp operon contains the Tryptophan biosynthetic
  genes.
• Trp repressor protein can bind to the operator of
  Trp operon
• When tryptophan is high, it binds to the
  repressor and induces a change so that the
  repressor can now bind to DNA.
• When tryptophan are low in the cell, tryptophan
  falls off the repressor, and the repressor goes
  back to its original conformation, losing its
  ability to bind to the DNA. RNA polymerase binds
  to the promoter and transcription proceeds,
  making tryptophan biosynthetic genes and
  replenishing the cell's supply of tryptophan.
• This type of feedback inhibition of transcription
  is very common. ribosomal RNA can also act to
  repress their own synthesis.

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Repressible Operon Trp Operon

• Repressible Operon Operon that is usually ON
  but can be inhibited
• The Trp Operon
• example of a repressible operon
• Genes that code for enzymes needed to make the
  amino acid tryptophan

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TrpR Gene

• TrpR gene is the regulatory gene for the Trp
  operon
• Found somewhere else on the genome
• NOT part of the Trp operon
• TrpR gene codes for a protein TrpR repressor
• TrpR gene is transcribed and translated
  separately from the Trp operon genes.

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TrpR Repressor

• Repressor protein is translated in an inactive
  form
• Tryptophan is called a corepressor
• When tryptophan binds to the TrpR repressor, it
  changes it into the active form

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Operator Region

• There is also an operator region of DNA in the
  Trp Operon
• Just after the promoter region
• The TrpR Repressor can bind to the operator if
  its in the active form

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Trp Operon

• Transcription is ON
• Occurs when there is no tryptophan available to
  the cell.
• Repressor is in inactive form (due to the absence
  of tryptophan)
• RNA Polymerase is able to bind to promoter and
  transcribe the genes.

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Trp Operon

• Transcription is OFF
• Occurs when tryptophan is available
• Tryptophan binds to the TrpR repressor ? converts
  it to active form
• TrpR protein binds to operator ? blocks RNA
  Polymerase ? no transcription

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trp a repressible operon
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Question

• Under what conditions would you expect the trp
  operon to go from OFF to ON again?
• When there is no longer tryptophan available all
  of it has been used up

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The Histidine Operon (An Attenuator)

• The histidine operon functions in a slightly
  different way.
• At the beginning of the operon there is a leader
  coding region
• AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-
  CAU-CAU-CCU-GAC
• Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-
  His-His-Pro-Asp
• When transcription begins, the RNA comes of
  the DNA and ribosomes hop onto it to start
  translation.
• Low amount of histidine in the cell
• - the ribosome stalls because no aminoacyl
  tRNA's charged with histidine.
• - this leaves a long stretch of RNA (for
  RNA ploymerase is still transcribing
• it) with no ribosomes bound to it.

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The Histidine Operon (An Attenuator)

• High amount of histidine in the cell
• - the ribosome is not stalled
• - the leader sequence in RNA allows it to
  form a terminator loop
• (attenuation site), at which point the
  RNA is cleaved
• - RNA polymerase stops transcribing the
  genes
• - the terminator only functions when the
  ribosome is not stalled.
• Many amino acid synthetic operons are also
  controlled by some form of attenuation. The
  tryptophan operon has both attenuation control
  and repressor control.

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Methods for Studying Regulation

• Possible mutations in various elements of the Lac
  operon, give rise to mutants
• How to study the lac operon? Tools used
• IPTG (isopropyl-beta-D-thiogalactoside)
• - a molecule analogue to lactose, binds to
  the Lac repressor (Lac I).
• - used as a gratuitous inducer to induce
  Lac operon
• - but not a substrate for the lactose
  metabolism genes.
• Spectrophotometer quantification of
  B-galactosidase activity.
• - Quantify amount of mRNA made (coding
  lacZ, lacY, and lacA)
• - B-galactosidase can cleave a colourless
  substrate called ONPG into a
• yellow product , ONP - quantitated by
  spectrophotometer.
• - The degree of yellowness - indicates
  enzyme activity or amount of
• transcription of mRNA or the activity of
  lac operon.

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Methods for Studying Regulation

• Different types of gene expression
• - Constitutively ( c ) expressed gene is
  never turned off, it is
• making mRNA and protein all the time.
• - Inducible gene can be turned on by an
  inducer.
• - Uninducible gene is never turned on. DNA
  binding site is
• mutated preventing binding by an
  inducer.
• - Super-repressor ( s ) always represses,
  regardless
• of its regulation. eg. a Lac I (s)
  mutant always represses the
• promoter whether or not lactose is
  present.

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Cis or Trans-regulation

• DNA element 1 and DNA element 2
• How to determine whether DNA element 1 is
  acting in cis or in trans to one another ?
• Test insert a piece of DNA carrying DNA
  element 1 into a cell that already has a copy of
  mutated DNA element 1 adjacent to DNA element 2.
• A) Observation the cell recovers its
  function.
• Conclusion The inserted element can
  complement or replace the function of the mutated
  element 1, it can be said to be trans acting,
  since it must diffuse off a plasmid or from
  another site in the DNA in order to be
  functional. This, therefore involves a diffusible
  protein.
• B) Observation the cell does not regains
  its function
• Conclusion the two functional pieces of
  DNA must be adjacent to each other to be
  functional (cis acting),

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BLA OPERON- Control of gene expressing
resistance to Penicillin

• Usually present on a plasmid in S aureus
• its function is B-lactam-induced production of
  penicillinase.
• Bla operon composed of 3 genes
• blaZ codes for a penicillin-hydrolysing
  enzyme (penicillinase)
• blaR1 bla l transcription regulator
  genes
• When penicillin is in the environment,
  membrane-bound signal transducer protein BlaR1
  recognises it and transmits the signal to the
  cytoplasm.
• Repressor protein Bla I, which binds near to the
  promoter of blaZ preventing its transcription, is
  cleaved off.
• blaZ is transcribed efficiently to produce
  penicillinase.

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Negative regulation Substrate induction
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Positive regulation of the lac operon
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Positive Gene Regulation

• In the lac operon there are other molecules to
  further stimulate transcription.
• Lactose will only be digested for energy when
  there isnt much glucose around
• When glucose levels are low, level of cAMP
  molecule builds up

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cAMP and CAP

• CAP regulatory protein that binds to cAMP
• CAP is inactive unless cAMP binds to it

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Positive gene regulation

• If there isnt much glucose? high levels of cAMP
• CAP and cAMP bind ? CAP can bind to the promoter
  ? stimulates RNA Polymerase to bind

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Positive gene regulation

• When glucose levels rise again, cAMP levels will
  drop ? no longer bound to CAP
• CAP cant bind to promoter ? transcription slows
  down

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Positive gene regulation

• The lac operon is controlled on 2 levels
• Presence of lactose determines if transcription
  can occur
• CAP in the active form determines how fast
  transcription occurs

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Lac Operon
Operon is on and enhanced
Operon is ready to be enhanced but is shut off
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Lac Operon
Operon is off and un-enhanced
Operon is on but un-enhanced
POWERade is a drink manufactured by The Coca-Cola
Company.
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Lac Operon
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Induction by negative or positive control
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Negative regulation end-product repression
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Do all operons have operator regions?

• NO
• There are some genes that always need to be
  transcribed ? they do not need to have operators
  to regulate them in this manner.
• Ex. genes that participate in cellular respiration

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Thank U