The LAC Operon

1
The LAC Operon

• Operon A set of adjacent genes whose mRNA is
  synthesized in one piece, plus the adjacent
  regulatory signals that affect transcription of
  the genes.
• Types of operons
• Inducible substrate needs to be present before
  transcription of genes involved in its breakdown
  occurs. Mostly metabolic pathways - breaking
  things down for energy e.g. lac operon
• Repressible anabolic pathways (building things)
  no reason to make protein to build a molecule
  that is already available. e.g. Tryptophan
  operon. An E. coli cell wont waste a bunch of
  energy making tryptophan if it is available from
  the medium. Tryptophan represses its own
  synthesis.

2

• Lac Operon
• -This operon is inducible by allolactose (a
  modified lactose)
• -A cluster of genes coding for proteins in the
  lactose metabolism pathway
• -Coordinated transcriptional regulated genes
• -One common promoter, repressor and activator
• -System less complex by reducing the number of
  genes needed to regulate it
• -When lactose is present a polycistronic mRNA is
  produced containing all the genes needed to
  metabolize lactose

3

• Lac Operon Structure (Fig 14-3 in text)
• Ppromoter lacAtransacetylase
• Ooperator lacIrepressor
• lacYpermease lacZß-galactosidase
• CAPcatabolite activator protein
• lacYtransmembrane protein allows lactose to
  enter cell
• lacA-function unknown
• CAPcan repress lac operon as part of a general
  sugar metabolism program

4

• Two forms of regulation
• 1) cis regulation of transcription (cis
  adjacent)
• refers to DNA sequences that regulate the
  transcription of downstream genes, through
  interaction with DNA binding proteins
• Promoter
• RNA polymerase II binds to promoter at a sequence
  called the TATA box
• Initiates transcription of the lac genes
• Operator
• Binds a repressor which inhibits DNA synthesis
• Overlaps with the promoter by about 9 bp
• CAP binding sites
• Within the promoter sequence
• Activated CAP binds to this sequence and results
  in more efficient binding of RNA polymerase II
  (increases binding 1000 fold).
• Turns the operon ON
• Without CAP bound there is a very small amount of
  the lac genes produced still referred to as
  OFF
• The promoter CAP binding sites are considered to
  be positive cis elements.
• Function in activating the operon.
• The operator is a negative cis element.
• Function in deactivating the operon

5

• Two forms of regulation
• 2) Trans regulation of transcription (trans
  across)
• Proteins that regulate transcription by binding
  to cis elements
• a) Repressor
• Has two binding sites
• One is a DNA binding site, associates with
  operator as a tetramer
• Inhibits RNA synthesis by blocking the action of
  RNA polymerase II
• Forms a large complex next to the promoter and
  doesnt allow enough space for the polymerase to
  bind called STERIC HINDERANCE
• When the inducer (allolactose) binds the
  repressor (lacI) it undergoes a conformational
  change and can no longer bind the operator as a
  tetramer. Allows RNA polymerase II to bind.
• Modification of conformation due to the binding
  of a small molecule ALLOSTERIC REGULATION
• b) RNA polymerase - Transcribes the RNA
• c) CAP - Increases RNA polymerase binding
  efficiency requires cyclic AMP to be active

6

• Bacterial Cell Physiology-E. coli in the human
  intestine
• Cells prefer glucose to any other sugar,
  including lactose.
• given the choice all glucose is used first
• metabolic pathways (energy making) are tuned in
  all organisms to use glucose
• all other sugars require modification either into
  glucose or some intermediate in the glycolytic
  pathway. This takes energy and makes the process
  less efficient.
• the lac operon will be off if glucose and
  lactose are available
• Repressor is bound when glucose is present, CAP
  is inactive
• A small amount of leaky transcription occurs.
  Just enough permease and ß-galactosidase is
  produced to allow lactose into the cell to be
  converted to allolactose to activate the system
  if glucose runs out.

7

• Bacterial Cell Physiology (cont)
• if the cell runs out of glucose it will switch to
  lactose and other sugars
• this switch is modulated by cyclic AMP
• cAMP is required to activate CAP and turn the
  system ON
• AMP is the signal for the cell that is running
  low on energy. All the energy of the cell is
  contained in the two phosphate bonds of ATP
• if the cell has lots of glucose the AMP is
  converted to ATP. As long as the cell is
  producing ATP no AMP is accumulated
• glycolysis
• AMP ATP
• metabolism
• If the cell runs out of glucose metabolism slows
  down, AMP accumulates. The cell doesnt have
  enough energy to turn it all back into ATP.
• Small amount so AMP are converted to cyclic AMP
  which activates the CAP protein and allows the
  cell to produce enzymes needed for the breakdown
  of alternative sugars available in the
  environment.
• cAMP AMP ATP
• adenylyl cyclase
• Referred to as catabolic repression glucose
  represses cAMP levels, CAP protein is inactive
  and transcription of lac genes is OFF

8

• Partial Diploid Systems
• Used to define were the cis and trans acting
  components of the operon are
• F plasmids (normally involved in mating
  response) were constructed containing all of the
  components of the lac operon and transformed into
  E. coli cells. And the genes were modified to
  see what role they played in the system.
• Remember
• Cis factors DNA sequence that affects only
  downstream genes
• Trans factors proteins that affect DAN binding

Z
Y
Z
Y
O
O
A
A
P
P
F Plasmid
Bacterial Chromosome
9

• Structural genes
• One functional copy of a gene lactose is
  metabolized
• Two non-functional copies of a gene present in
  the cell leads to a non-functional metabolic
  pathway
• Remove lacY lactose cannot enter the cell
• Remove lacZ lactose cannot be broken down into
  glucose and galactose
• Operator
• Normal repressor can bind (O)
• Mutated constitutive expression negative
  regulation (expressed as O- or Oc)
• Repressor cant bind so the operator is
  desensitized to the lacI protein
• Transcription occurs whether lactose is present
  or not
• Promoter
• Normal RNA polymerase can bind (P)
• Mutated RNA polymerase never binds (P-)
• Inhibitor
• Normal can bind the operator when no lactose is
  present. (I)
• Two mutants
• 1) Mutated DNA binding domain never binds
  operator, operon is not sensitive to inhibition
  by lacI protein. (I-)
• 2) Mutated allolactose binding site (Is
  super suppressor) loses the ability to associate
  with allolactose the tetramer can never be
  removed from the operator in response to lactose
  entering the cell

10

• EXAMPLE (assume CAP is activated unless otherwise
  stated)
• A) IPOcZY-A/I-POZYA
• B) IP-OcZ-YA/I-POcZY-A
• C) IPOZYA/ISPOcZYA
• D) I-P-OZYA/I-POcZY-A
• E) ISPOcZ-YA/IP-OcZ-Y-A
• Permease ß-galactosidase
• no lactose lactose no lactose lactose
• A -
• B - -
• C
• D - -
• E - -