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MCB2023S > The lac operon > Flashcards

The lac operon Flashcards

1
Q

Why are mutations important?

A
  • Mutations are important tools of genetic analyses.
  • Key principles and concepts in genetics have been obtained through analyses of mutants.
  • Mutations allow fundamental biological processes to be dissected so that the overall biological system can be understood
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2
Q

What are the 6 proofs of the Lac Operon Model?

A
  1. Genes transcribes on single mRNA transcript
  2. Allolactose and not lactose is the inducer
  3. lacI encodes a repressor
  4. Prove roles of cis and trans-acting elements
  5. LacI binds to Operator region in promoter
  6. LacI binds to inducer molecule
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3
Q

What is the different between an operon and a regulon?

A
  • Operon:
    o Group of genes physically linked to the chromosome under the control of the same promoter which gives rise to a polycistonic mRNA.
  • Regulon:
    o Group of genes needed for the same process but located in different parts of the chromosome and containing their own promoter, but the promoters are regulated in the same fashion to allow for coordinated expression.
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4
Q

Structure of the Lac Operon

A
  • plac = Promoter for structural genes
  • pi = Promoter for regulatory gene
  • lacI = Regulatory gene codes for repressor protein
  • lacZ = structural gene for B-galactosidase
    o Converts lactose to glucose + galactose
    o Minor reaction = converts lactose to allo-lactose
  • lacY = structural gene for B-galactosidase permease
    o Transports lactose into cell
  • lacA = structural gene for B-galactosidase transacetylase
    o Function not known, and not super required.
  • Polycistronic operon
    o 1 promoter and 1 regulator transcribing 1 mRNA containing several genes; translated into many proteins – allows coordinated induction/repression.
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5
Q

Regulation of the lac operon

A
  • Default state of inducible operon is to be OFF
  • Negative control:
    o Regulated by lac repressor protein which binds to operator site in presence of glucose
  • Positive control:
    o Regulated by catabolite activator protein (CAP) which binds to the CAP site via cAMP in the absence of glucose
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6
Q

Positive Regulation of the lac operon

A
  • Lactose but no glucose
  • No glucose = No inactivation of adenylate cyclase
    o ATP produced; cAMP produced
    o CAP binds to cAMP and becomes activated
    o Binds to CAP site and enhances transcription.
-	Lactose present: Allolactose produced
      o	Inducer (allolactose) inactivates Repressor so RNA Pol can bind
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7
Q

Negative Regulation of the lac operon

A 
-	Glucose but no lactose:
      o	Inducer (Allolactose) absent because no lactose, so repressor remains bound, blocking RNA Pol
      o	Glucose means adenylate cyclase is inactive so no cAMP is formed, so CAP doesn’t bind to CAP site
  • Neither Glucose nor Lactose:
    o cAMP is formed, so CAP binds to CAP site
    o Allolactose still not formed so repressor remains active and blocks RNA Pol
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8
Q

Catabolite Repression

A
  • Both Lactose and Glucose:
    o Allolactose produced, repressor inactivated
    o Glucose present means adenylate cyclase inactivated so no cAMP is formed, so CAP is not activated
  • Doesn’t mean there’s no transcription, just very very little.
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9
Q

Trans-acting control elements

A
  • LacI- repressor protein
    o Physically linked to Lac operon but transcribed independently of lacZYA and binds to lacO.
  • Catabolite Activator protein
    o Located on a different part of the chromosome and transcribed independently of lacZYA
    o Must bind to cAMP before binding to CAP site.
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10
Q

Cis-acting control elements

A
  • lacP
    o Promoter region – binding site of RNA Polymerase (-10 & -35 sites)
  • lacO
    o Operator region – binding site of Lac repressor
  • CAP site
    o Binding site of CAP + cAMP
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11
Q

Induction of the lac operon

A
  • Inducible - Operon repressed
  • Lactose taken up via permease (LacY)
  • Converted to allolactose (lactose isomer) by - β galactosidase.
  • Interacts with bound Lac repressor protein - allosteric modification of repressor so it can no longer bind to lacO.
  • No longer interferes with RNA polymerase binding to lacP
  • Transcription of operon occurs.
  • Gene products produced at high levels till lactose depleted.
  • Inducing signal drops
  • Lac repressor no longer inactivated (because allolactose not being produced anymore) binds to lacO
  • Operon repressed
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12
Q

Proof that lac operon is polycistronic

A
  • Created DNA probes specific for lacZ, lacY, lacA and would bind to RNA molecule
  • You would expect: if the probe binds to 3 separate mRNA, you would have 3 bands in electrophoresis.. if polycistronic you would have 1 band
  • Northern Hybridization was used –
    o RNA extracted
    o Electrophoresis separates RNA by size
    o Transfer RNA onto membrane
    o Fix it there with UV or heat
    o Allow labelled DNA probes to bind to RNA
    o Visualization of labeled bands on X-ray film
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13
Q

Proof that allalactose and not lactose is the inducer

A

Experiment 1: Use a lacZ- lacY+ mutant
- Can transport lactose (lacY+) (still has permease +ve)
- LacZ is non-functional (but still there) - Cannot produce allolactose or use lactose (B-galactosidase -ve)
- Grew on 2 complete media without glucose; 1 had lactose, 1 didn’t.
- Assayed for increase in transcription of lac operon
o Gene is nonfunctional but mRNA is still being made
o IF lactose was inducer, then its presence should result in induction of lac operon and increase in transcription.
- Found that mRNA levels are the same in the presence or absence of lactose
- THEREFORE: lactose is not the direct inducer

Experiment 2: Use lacZ- lacY+ mutant

  • Answering question = Could one of the breakdown products of the lactose by lacZ be the inducer?
  • Repeated same experiment using different sugars (lactose, allolactose, glucose, galactose) in media
  • Lac operon was only transcribed in response to allolactose
  • CONCLUSION: Only allolactose induced transcription
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14
Q

Proof that lacI encodes a repressor

A
  • Create a lacI mutant: lac i- z+ y+
    • i- = nonfunctional LacI repressor is made (won’t bind to O region and block transcription)
  • Compared with wild type: lac i+ z+ y
    • i+ =Active functional LacI repressor made
  • Performed growth assay on wild-type and lacI mutant:
    o Add lactose at T = 0
    o Measure B-galactosidase protein amount versus time
    o Add allolactose analogue, Isopropyl-B-D-thiogalactoside (IPTG) when lactose depletes.

Results:
- B-galactosidase produced continually with or without the inducer

  • Reasoning:
    o Wild-type:
    • Permease takes up lactose and B-gal converts to allolactose
    • B-gal amount increases and after ±90mins it stops when lactose used up
    • If IPTG added again, B-gal amount increases

o LacI mutant:

- In LacI mutant – constitutive production of B-galactosidase in presence or absence of inducer
- Therefore lacI encodes a repressor (LacI)
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15
Q

What is the most important proof of the lac operon in terms of determining its structure and function?

A

Proof of cis and trans acting elements

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16
Q

Proof of trans acting element

A

Experiment 1:
- F’ LacZ+ LacI- LacO+/LacZ- LacI+ LacO+
- Meaning that LacZ transcription happens in the plasmid (non-functional in the chromosome). If LacI was trans-acting, it should be able to diffuse from chromosome and bind to both LacO sites and prevent LacZ transcription.
- LacI+ LacZ-/LacI- LacZ+ produces B-gal only in presence of lactose because it inactivates the repressor
- Proves that the lacI gene is trans-dominant
o Overall result: the phenotype of this strain will be same as wild-type

Experiment 2:
- LacIS produces a super repressor that cannot bind to allolactose and remains inactivated in the presence of an inducer.
- LacIS super repressor remains bound to LacO site
- In presence of lactose: LacI super repressor remains bound to LacO because it can’t bind to allolactose
- LacIS LacZ+/LacI+ LacZ+ will not produce B-gal in the presence or absence of lactose as LacIS is trans-dominant.
o Overall result: this strain will produce NO LacZ protein in presence or absence of lactose.

17
Q

Proof of cis acting element

A

Experiment 1:
- F’ LacZ+ LacI+ LacOC / LacZ- LacI+ LacO+
- Mutations in sequence of operator site of lac operon prevent LacI binding – LacOC
- In the absence of lactose, LacI repressor can bind to LacO on the chromosome but not to the LacOC on the plasmid
o B-gal produced from plasmid.
- In the presence of lactose, LacI repressor protein cannot bind to the chromosomal LacO, but neither to the LacOC
o B-gal produced.
- LacOC is dominant over LacO: Mutations in lacO are constitutive and cis-acting.
- Overall result: LacZ will be produced in presence and absence of lactose.

Experiment 2:
- Chromosome:
o LacZ+
o LacI+
o LacO+
- Plasmid:
o LacZ-
o LacI+
o LacOC
- LacO only affects genes to which it is physically connected to.
- LacOC cannot prevent LacI from binding to functional LacO.
- In the absence of lactose, LacI repressor can bind to LacO on the chromosome and repress transcription
o No B-gal produced.
- In the presence of lactose, LacI repressor protein cannot bind to the chromosomal LacO, but neither to the LacOC
o B-gal produced.
- Overall result: the phenotype of this strain will be same as wild-type.

18
Q

Proof lacI repressor binds to O region in promoter

A
  • LacI purified
  • Run lacO DNA on gel shift with different repressor proteins
  • Perform DNAse footprinting technique
  • Findings:
    o LacO is palindromic - allows repressor to bind as dimer
    o LacOc mutation
    o LacIs super repressor
    o LacITB Tight Binding Repressor
    o Multiple O sites
19
Q

LacOc Mutations

A

o LacOc- Constitutive mutants
o Specific DNA base pair mutations in the lac operator that prevent proper contact of the DNA-binding domain of Lac repressor will result in lacOc mutants – LacI cannot bind to lacOC

20
Q

LacIs Super repressor

A
  • LacIs = uninducible mutants
  • Always bound to the operator, always preventing transcription
  • There are two possible causes for lacIS mutations:
    o Repressor cannot bind to the inducer (allolactose/IPTG) but can bind to lacO and remain bound
    o Repressor can bind to inducer but mutations in the LacIS regulatory domain (inducer binding domain) prevents LacIS from changing conformation in the presence of inducer and releasing itself from being bound to lacO (does not fall off of the operator)
  • Uninducible phenotype, the super repressor mutation (lacIS) is dominant to wild type.
  • Once lacIS is bound to LacO, it will always be bound so the wild-type repressor will not cause the IS repressor to fall off the operator.
21
Q

LacItb Tight Binding Repressor

A
  • lacItb repressor can bind inducer and can undergo a conformational change but cannot be released from lacO
    o There is a mutation in the binding region of LacItb that allows only to bind and not be released from lacO
    o It is important to note that IacItb is different from IacIS because the mutations in these genes affect different domains of the lac repressor protein.
  • Uninducible phenotype, IacItb is dominant to wild-type.
22
Q

LacO multiple Operator sites

A

o The main lac operator site is O1 which is adjacent to the promoter

o There are auxiliary operators upstream (lacO3) and downstream (lacO2) of lacO1

o All lac operators are required for optimal repression

o Lac repressor binds lacO1 and either one of the other operator sites (lacO2 or lacO3) to block transcription.

o This is done by tetramerization of Lac repressor dimers whereby it allows formation of a DNA loop

o The DNA loop between O1 and O3 will contain the -35/-10 binding sites (promoter) and CAP site, so these sites become relatively inaccessible to both RNA Polymerase and CAP protein respectively which in turn prevents transcriptional initiation.

o The DNA loop between O1 and O2 will exclude part of the lacZ gene, the promoter and CAP site - RNA polymerase and CAP can bind but the loop will prevent RNA polymerase from elongation.

23
Q

Proof that LacI binds to inducer molecule

A 
Technique = Equilibrium Dialysis
Dialysis = technique of protein purification using differences in pore size and molecular weight
  • Time = 0 min
    o LacI protein in dialysis bag (pore size small enough that LacI can’t leave)
    o Solution of allolactose outside
  • Time = Equilibrium
    o LacI protein in bag
    o Solution of allolactose more inside than outside
    o Has bound to LacI protein
    o Complex too large to diffuse out (if it wasn’t bound to LacI it would be able to freely diffuse out)
  • Equilibrium control:
    o Using LacI and other compound known not to bind to LacI.
    o LacI protein in bag
    o Solution of compound* equal concentration inside and outside
    o Protein has not bound the compound
    o Compound continues to diffuse in and out = equilibrium
24
Q

Glucose control of cAMP levels

A
  • High Glucose:
    o Low Adenylate Cyclase activity = low cAMP levels
  • Low Glucose:
    o High Adenylate Cyclase activity = high cAMP levels
25
Q

Glucose control of Adenylate Cyclase activity

A 
-	Glucose absent:
	o	Glucose enters via PTS system
	o	IIAglu- P means IIA is phosphorylated
	o	Phosphorylation of IIA increases enzyme activity of adenylate cyclase activity
	o	Leads to conversion of ATP into cAMP
  • Glucose present:
    o Transported into cells via IIC
    o Phosphate on IIA is added to glucose -> glucose-6-P which allows glucose to be broken down in the cell
    o Because IIA isn’t phosphorylate, theres no/very little enzyme activity of adenylate cyclase.
26
Q

CAP regulation of lac operon

A
  • CAP-cAMP-DNA complex formed
  • Specific binding sites in promoter region – CAP site
  • Enhances binding of RNA polymerase activity at promoter
  • Increased transcription of operon
  • CAP is a positive Transcriptional Regulator:
    o CAP + cAMP interacts with RNA polymerase subunits
    o Improves recognition and transcription by RNA polymerase
27
Q

Proof that CAP binds cAMP for positive regulation of lacZ

A
  • Grow E.coli (lactose only) with mutation in CAP that causes it to bind cAMP weakly
  • Increase the concentration of cAMP and measure lacZ expression

MCB2023S (9 decks)

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