a l o c t s e ( I P T G ) l a c I P O l a c Z l a c Y l a c A c r p A R N P o l . A M P C y

R e g S t r I R I R

Induction Positive Negative Loss of function DNA binding Effector binding (total) Positive Induction Repression Negative Non inducible Non de-repressible Non inducible Non de-repressible Non inducible De-repressed Constitutive De-repressed Constitutive De-repressed Non inducible (super-rep.) De-repressed

+: inducible or repressible c/d: constitutive or derepressed Positive Negative R+/R- + R+/Rc/d c/d Rc/d/R- c R+/R- - R+/Rc/d + Rc/d/R- - +: inducible or repressible c/d: constitutive or derepressed -: non expressed (non inducible, non derepressible)

lacI+/lacI-(c) lacI+/lacIs(ni) lacIs(ni)/lacI-(c) inducible Non-inducible (superepressed) lacI+/lacIs(ni) lacIs(ni)/lacI-(c)

nirA NO3- niiA niaD crnA areA NH4-

nirA+ 1 100 nirA- 1 1 nirA+/nirA+ 1 100 nirA-/nirA- 1 1 niiA niaD nirA+ 1 100 nirA- 1 1 nirA+/nirA+ 1 100 nirA-/nirA- 1 1 nirA+/nirA- 1 100 nirA+/nirA- 1 1 nirA+/nirA- 1 50 S S

c nirA+ 1 100 nirAC 100 100 nirA+/nirA+ 1 100 nirAc/nirAc 100 100 nirA+/nirAc 100 100 nirAc/nirA+ 1 100 nirA+/nirAc 50 100 c c c

Other criteria: Phenotype of complete loss of function-how to identify them Frequency of mutations Phenotype and frequency of revertants of  « - » chain termination and deletion mutants in positive c/d in negative c/d< or << than - c/d> or>> than - reverts rarely to + -reverts frequentely to c (or d)

Two types of mutations: alkalinity mimicking acidity mimicking pacC Two types of mutations: alkalinity mimicking acidity mimicking Alkaline pH, products of pal genes proteolysis alkalinity expressed acidity expressed

Induction Positive Negative Loss of function DNA binding Effector (total) binding Positive Induction Repression Negative Non inducible Non derepressible De-repressed Constitutive (super-rep.) Constitutive De-repressed Non-inducible (super-rep.) Non-derepressible « Real » gain of function

Induction Positive Negative Loss of function DNA binding Effector binding (total) Positive Induction Repression Negative Non inducible Non inducible Non inducible De-repressed Constitutive De-repressed Constitutive De-repressed Non inducible (super-rep.) De-repressed

active inactive 1/16 4/16 6/16 4/16 1/16 (1/4) (1/4) active ? ? ? inactive

O1 O2 O1 O2

M1 M2 + M1 M2

l a c I P O1 l a c Z l a c Y l a c A O2 O3 96bp 401bp

a l o c t s e ( I P T G ) l a c I P O l a c Z l a c Y l a c A c r p A R N P o l . A M P C y

+ O c R S 1 2 + - O c R S 1 2

+ O c R S 1 2 + - O c R S 1 2

const. for S2; inducible for S1 const. for S1; inducible for S2 + - O

R S (IS) P O S1(Z) S2(Y) S3(A) R S (IS) P OC S1(Z) S2(Y) S3(A)

+ - O c R S 1 2 + - O c R S 1 2 "

S2 S1

S2 S1

S2 S1 S2 S1 S2 S1 S2 S1

GAL10 niaD GAL1 niiA

Elf et al., 2007

dilution plating Escherichia coli low IPTG (about 0.1 M) low IPTG+ X-GAL Escherichia coli low IPTG (about 0.1 M) dilution plating Novick and Weiner, 1957

Choi et al., Jan 2, 2009

Jacob, F., and Monod, J., Genetics regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 1961, 3, 318-356, reprinted in Taylor, J. H., Selected papers on Molecular Genetics, 1965, Academic Press Ptashne, M., A genetic switch: Phage land higher organisms, 1992 Cell press and Blackwell house (there is a 2004 edition). Muller-Hill, B., The lac operon, 1996, de Gruyter Ptashne, M.and Gann Alexander, Genes and Signals, Cold Spring Harbor, 2002