PM-251 lecture 2

• The Operon Concept


  Where did it come from?
• Francois Jacob, Jacques Monod & André Lwoff


  Institut Pasteur, Paris
• Sugar metabolism in E. coli

  1. Hydrolysis of the disaccharide Lactose (from milk)

  2. Enzyme Induction: new β-Galactosidase enzyme is only produced when its substrate, lactose, is present in the culture medium

  3. Lactose was called an Inducer of the enzyme β-Galactosidase
• Normal or "Wild Type" (wt) cells

  • + Lactose → Enzyme produced
  • - Lactose → NO Enzyme produced
• Inducible


  Wt cells show enzyme induction
• Constitutive mutant cells


  • + Lactose → Enzyme produced
  • - Lactose → Enzyme produced
• Constitutive


  Mutant cells show continuous enzyme production regardless of lactose (as a part of their "constitution")
• Inducibility gene, i


  In wt cells this would be normal, i+, in constitutive mutants, somehow defective: i-
• All the constitutive mutant colonies were compared after the location of their mutant gene was determined by genetic mapping
• The mutant genes mapped in two loci not one. Therefore, there must be at least TWO different genes affecting inducibility, rather than the one gene called i
• o


  The second "gene" was eventually called o. In wt cells this would be normal, o+, in constitutive mutants, somehow defective: o- or oc (superscript c for constitutive)
• Complementation tests


  1. Can the various mutants and wild type compensate for their defects if they are crossed with each other

  2. Bacteria are haploid – no (normal) sex – no Mendelian experimental crosses: for complementation experiments the second copy of a gene has to be carried into a cell on a plasmid

  3. The F' episome or Fertility plasmid was used to carry the test genes

  4. The recipient cell then becomes diploid only for the genes in common on the plasmid and chromosome – not for all the genes on the bacterial chromosome – a micro-diploid or partial diploid or merodiploid
• β-galactosidase gene, z


  In wt cells this would be normal, z+. (Mutants, z-, which are somehow defective in forming an active enzyme structure were also found.)
• i+ z+ cells


  INDUCIBLE (wt)
• 1. z+ cells


  CONSTITUTIVE (mutant)
• i+ is dominant to i-

  1. is recessive to i+

  i+ can complement i-
• o+ z+ cells


  INDUCIBLE (wt)
• oc z+ cells


  CONSTITUTIVE (mutant)
• oc is dominant to o+

  o+ is recessive to oc

  o+ cannot complement oc
• The great leap forward – a hypothetical model: the operon concept
• Operon concept


  1. TRANS-ACTING LOCUS ("gene") synthesizes a protein ("?RNA?" - Monod) molecule = REPRESSOR PROTEIN which specifically binds to CIS-ACTING LOCUS = BINDING SITE ON DNA = OPERATOR SITE

  2. REPRESSOR PROTEIN bound to OPERATOR DNA blocks access of RNA Polymerase to PROMOTER: transcription is turned off

  3. Small molecule INDUCER (IND) binds to REPRESSOR protein (R) and prevents REPRESSOR binding to OPERATOR DNA

  4. RNA Polymerase can now access PROMOTER: Structural Genes transcribed (& translated), enzymes expressed
• The gene labelled R for Repressor in this scheme had already been called i or I (for inducibility) before the Lac Operon concept was developed: it was then "too late" to give it the more logical label "R"
• Cis-acting


  The influence of a gene is confined to its own DNA molecule
• Trans-acting


  A gene can synthesize a protein that can influence genes on a different DNA molecule
• Repressor protein


  A protein that specifically binds to the operator DNA and blocks access of RNA polymerase to the promoter, turning off transcription
• Lac operon regulation


  1. Repressor protein binds to operator DNA

  2. Repressor protein dissociates from operator DNA when inducer binds to it

  3. RNA polymerase can now access promoter and transcribe structural genes
• The gene coding for the repressor protein was originally called i or I (for inducibility) before the lac operon concept was developed
• The small molecule inducer (lactose) should not be confused with the i gene coding for the repressor
• The repressor gene can be on the same DNA molecule as the structural genes or on a different DNA molecule
• i+ allele


  Produces a normal repressor protein that can be inactivated by an inducer
• is mutant


  Produces an altered repressor protein that cannot bind the inducer, resulting in super-repression
• iq mutant


  Maps in the promoter of the repressor gene and overproduces the normal repressor protein
• Allo-lactose


  A structural isomer of lactose produced by the β-galactosidase enzyme, which is the natural inducer of the lac operon
• The lac promoter is naturally "leaky" to allow some transcription of the structural genes even in the absence of lactose
• Gratuitous inducer


  An inducer that cannot be a substrate for the enzyme it induces
• Gratuitous inducers


  • IPTG (Isopropyl-β-D-thiogalactopyranoside)
• Chromogenic substrate


  A substrate analogue that releases a coloured product upon hydrolysis by an enzyme, allowing easier detection and assay
• Chromogenic substrates


  • ONPG (ortho-Nitrophenyl-β-D-galactopyranoside)
  • X-Gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
• Fluorogenic substrate


  A substrate analogue that releases a fluorescent product upon hydrolysis by an enzyme, allowing more sensitive detection
• Fluorogenic substrate


  • MUG (4-Methylumbelliferyl-β-D-galactopyranoside)