Lac Operon: Gene Regulation

Lac Operon

• The lac operon provides a classic example of gene regulation
• Francois Jacob and Jacques Monod won the 1965 Nobel Prize in Medicine for their work on the lac operon

Lac Operon Components

• The lac operon consists of several key components:
• lacI: encodes a lac repressor which regulates the operon
• lacO: the operator region where the repressor binds
• lacZ: encodes beta-galactosidase, which breaks down lactose into galactose and glucose
• lacY: encodes permease, facilitating lactose uptake by cells
• lacA: encodes transacetylase, which helps manage toxic galactose derivatives

Basic Regulatory System: No Lactose

• In the absence of lactose:
• The lac repressor binds to the operator
• Transcription is inhibited, with very low levels of beta-galactosidase produced (less than 5 molecules per cell)

Basic Regulatory System: Lactose Present

• In the presence of lactose:
• Lactose is converted to allolactose (inducer)
• Allolactose binds to the repressor, inactivating it
• Beta-galactosidase production begins within 2-3 minutes
• Soon, approximately 5000 molecules of beta-galactosidase are produced per cell

Allolactose-Mediated Induction

• Allolactose induces expression of the lac operon by inactivating the lac repressor
• The lac promoter is only a modest match to the consensus sequence:
  • 4/6 base pairs match in the -35 region
  • 3/6 base pairs match in the -10 region, resulting in only modest levels of expression

Mutations and Constitutive Expression

• Inactivating the lacI repressor or the operator leads to constitutive expression
• Types of mutations:
  • Nonfunctional lacI: allows expression of the lac genes
  • Nonfunctional lacO: Prevents repressor binding, allowing expression of the lac genes

Partial Diploids -Acting Factors

• Partial diploids reveal the nature of regulatory factors:
• lacI is a trans-acting factor, meaning it is diffusible and can affect genes on different DNA molecules
• lacO is a cis-acting factor, affecting only genes physically linked to it

Lac Repressor Structure

• The lac repressor has a helix-turn-helix (HTH) DNA binding domain
• Most HTH proteins bind as dimers to inverted repeats

Additional Binding Sites

• Lac has two more, weaker binding sites for lacI
• Each contains an inverted repeat that can bind both sites on a lacI dimer
• Each lacI is a tetramer and can bind both repeats on O₁ and both on O₂ or O₃
• lacO₁ alone results in 100-fold repression of lac-Z
• A complete system results in 1000-fold repression

Protein-DNA Interactions Specificity

• Without inducer:
  • the operator has an equilibrium constant of 2x10^13 with 1 binding sites
  • other DNA has an equilibrium constant of 2x10^6 with 4 x 106 binding sites
  • leads to Spcificity (10^7)
• With inducer:
  • operator has an equilibrium constant of 2x10^10 with 1 binding sites
  • other DNA has an equilibrium constant of 2x10^6 with 4 x 106 binding sites
  • leads to Specificity (10^4)
• The normal level is approximately 20 repressor presents per cell

Catabolite Repression

• E. coli preferentially uses glucose from a mixture of sugars
• cAMP Receptor Protein (CRP)
  • Adenyl cyclase converts ATP to cAMP
  • Phosphodiesterase converts cAMP to AMP
  • High glucose leads to low cAMP levels
• Without cAMP = CRP has no effect
• With cAMP = CRP activates the Lac operon by stabilizing polymerase binding

Diauxic Shift

• In the absence of glucose, cAMP levels rise, activating CRP, which compensates for the poor lac promoter consensus sequence and increases gene expression by approximately 50X

Lac Operon States

• Glucose high, cAMP low, lactose absent: No gene expression (OFF)
• Glucose low, cAMP high, lactose absent: No gene expression (OFF)
• Glucose high, cAMP low, lactose present: Low level of gene expression (ON)
• Glucose low, cAMP high, lactose present: High level of gene expression (ON)

Lac Operon in Biotechnology

• The lac operon is widely used in biotechnology to clone and express genes
  • LacZ is easily detected via X-gal cleavage, which produces a blue color
  • Cloning is done by inserting a gene of interest (YFG) into a polylinker in lacZ
  • White colonies indicate a successful insert
  • Expression of YFG can be regulated with IPTG

Galactose Operon

• No galactose -Repressor binds
  • Allows pol binding but blocks open complex formation
• When galactose is present
• Changes conformation of Gal repressor
• Allows some transcription, but gal promoter is weak on its own
• In the absence of glucose
  • High cAMP CRP
  • High levels of gal expression

AraC Operon

• Functions to regulate the utilization of arabinose sugar in bacteria
• AraC Operon with Arabinose
• AraC Operon without Arabinose
• araC binds as a dimer to araO2 & aral₁ sites, but NOT aral2
• AraC dimer causes a bend in DNA
• Doesn't recruit RNAP, even if glucose is low
  • blocks CRP binding site?
• Ensures very tight regulation
• arabC binds as a dimer to aral, and aral2 → allows activation
• When glucose is low (cAMP high) CRP-cAMP binds just upstream → further activation
• Activation is believed to occur through recruitment of RNAP

Trp Operon

• Functions to regulate the production of tryptophan (an essential amino acid) in bacteria
• Trp operon when levels of tryptophan are very high
• Transcription initiation blocked by TrpR repressor
• Trp operon is further regulated by transcriptional attenuation
  • Mediated by a leader sequence encoding a peptide with two Tryp which can fold two different ways
• Takes advantage of the fact that bacterial mRNA can be translated as soon as is emerges from RNA pol
• Tryptophan present =No transcription of genes for Trp biosynthesis
• Tryptophan absent = Downstream genes transcribed
• RNA pol Terminator No Trp
• RNA pol Terminator Adequate Trp

SOS Response

• SOS Response: SOS genes are not on an operon, but are still coordinately regulated
• Lack of functional LexA allows SOS genes to be expressed by LexA Inactivation

Riboswitches

• Riboswitches can alter translation or transcription based on small molecule induced changes in RNA structure
• Translation riboswitch.
• Transcription riboswitch.
• Riboswitches also control some tRNA synthases
• The riboswitch controlling thiamine pyrophosphate is well characterized
• GmIS can distinguish between very similar metabolites

Ribosome Regulation

• Ribosomes make up ≈ 45% of bacterial dry weight and their synthesis consumes a large portion of cell resources
• Excess r-proteins block translation of r-protein mRNA
• Some r-protein operons are polycistronic, but blocking translation of first also blocks translation of the others
• Stringent response uncharged tRNA synthesis of ppGpp down regulation of rRNA and tRNA synthesis

Bacteriophage lambda (λ)

• Bacteriophage lambda (λ): Bacteriophage lambda (λ) and lysogeny
• Lambda can also integrate into the host chromosome and remain inactive indefinitely
• There are two cycles: lysogenic and lytic
• Lytic vs. lysogenic with more detail
• If conditions are not that favorable it may decide to become a prophage or replicate it's dna over and over again
• With enough stress with conditions the prophage comes to life and integrates back to the cell
• The text version of lambda has been over simplified.

Lytic Infection

• Lytic infection immediate early
• Lytic infection Delayed early : O & P DNA replication Q is an antiterminator for PR prime PR prime is constitutive, but terminates after 196 bp without Q
• Lytic infection Late A -J head and tail proteins S & R lysis proteins
• Note by this time, Cro will have turned of PL and PR Q is what causes transcription of downstream tail proteins

Lysogeny

Establishment of lysogeny lysogeny starts exactly the same way

• then deviates: cll & clll activate PE and PI Activate genes Make large amounts of cl àshut off PL & PR Activate PM Pl activates genes for integration into bacterial genome Because of high cl, cells will be immune to further a infection Maintenance is
• Cl repressor turns off PL & PR activates its own transcription from PM Lysogens are immune further infection by another lambda phage cl inactivates PL & PR of incoming phage

Lysogens

Induction of lysogenic phage

• in the lab :857 repressor inactive at 42C becomes active not estblish at LS
• after DNA damage to bacteria: DNA damage Increases single strand DNA DNA bound RecA cleaves lexA à induce SOS genes Can also cleaves cl (lambda repressor)

Regulatory Mechanisms

• Antiterminators: factors that prevent premature termination of transcription.
• These molecules bind to specific sequences on the RNA transcript known as "nut" sites This interaction allows RNA polymerase to bypass terminator sequences, allowing it to transcribe the entire genetic element.
• DNA binding = Transcription happens and there is protein present
• Repressors = DNA binds
• DNA does not bind and there is protein
• P is most important
• High C1 - Low CRO (good lysogen)

Lysogeny and Lytic infection battle

Lysogeny: ( Antisense for Q) activates, binds PL and PR,Shuts off cro and . cl activates PM

Activators

• Cll is rapidly degraded by hfl protease.
• hfl protease is inhibited by cAMP, and clll
• Low glucose increases high cAM increase high cl increase to lysogeny.

Retroregulation

• Retroregulation: Int is transcribed both by PL and PI Int protein is made only during lysogeny PL
• MRNA is degraded PI has a normal terminator and is relatively stable"""