Operon Theory

lacI: lac repressor

Lac operon and structural genes

lacI: lac repressorlacZ: β-galactosidase, LacY: β-galactoside permease and LacA: thiogalactoside transacetylase (also known as galactoside O-acetyltransferase) Lac repressorCatabolite activator protein(CAP)

Regulation by cyclic AMP

1. cya: adenylate cyclase which produces cyclic AMP

2. crp: catabolite activator or cAMP receptor protein(CRP)

Isopropyl-β-D-thio-galactoside (IPTG): It is not a substrate for β-galactosidase, so that it cannot be metabolized by E. coli

Phenyl-β-D-galactose (phenyl-Gal): is a substrate for β-galactosidase, but does not inactivate repressor and so is not an inducer

Lactose Analogues

ortho-Nitrophenyl-β-galactoside(ONPG): a colorimetric and spectrophotometric substrate for detection of beta-galactosidase

Allolactose (β1->6): isomer of lactose and the inducer of the lacoperon

Analogues

Classification of regulatory mutants: complementary test

lac Oc mutant, cis-dominant

lac I mutant

dominant

β-galactosidase transacetylase

Genotype -IPTG +IPTG -IPTG +IPTG Conclusion

I+Z+A+ <0.1 100 <1 100 Inducible

I+Z-A+ <0.1 <0.1 <1 100 lacZ encodes

β-galactosidase

I-Z+A+ 100 100 90 90 Constitutive

Table Phenotypes of repressor mutants

I-Z+A+ 100 100 90 90 Constitutive

I+Z-A+ /F' I-Z+A+ <0.1 100 <1 200 I+ >I- in trans

IsZ+A+ <0.1 <1 <1 <1 Noninducible

IsZ+A+ /F' I+Z+A+ <0.1 1 <1 1 Is >.I+ in trans

Trp operon and attenuation

Three enzymes required to convert chorismic acid into tryptophan:Three enzymes required to convert chorismic acid into tryptophan:1) Anthranilate synthetase

component I - encoded by trpEcomponent II - encoded by trpD

2) N-(5'-phosphoribosyl)-anthranilate isomerase/Indole-3-glycerol phosphate synthase - encoded by trpC

3) Tryptophan synthaseb-subunit - encoded by trpBa-subunit - encoded by trpA

trpL : coding for a short oligopeptide (trpL) which functions in attenuation.

trpR : Trp repressor protein . Unlike the lac operon, trpR is not adjacent to the operon. The structural genes are located at minute 28 on the E. coli map; trpR is located at minute 100. The TrpR repressor actually regulates the expression of two other operons in addition to the trpoperon.

TrpR protein is unable to bind to the operator by itself. As long as there is insufficient tryptophan in the cell, it will remain so. However, once the is insufficient tryptophan in the cell, it will remain so. However, once the level of tryptophan builds up, then the Trp repressor will block further transcription of the operon and, as a result, the synthesis of the three enzymes will decline.

Trp operon Attenuation

FIG. 1. Construction of pHGH807tacI containing the trp-lachybrid promoter tacI.