The lac operon An inducible operon Genes are

The lac operon – An inducible operon • Genes are either cis (coupling) acting – Genes affect those adjacent to themselves – Operator and structural genes Or • trans (repulsion) acting – Genes affect other genes that are not adjacent to themselves – Implies the action of a diffusible product – Regulator genes

Trans (repulsion) acting genes • Genes that affect other genes • Are not adjacent to the genes they control –Regulator genes Implies the action of a diffusible product

Genes that are cis (coupling) acting • Genes affect genes adjacent to themselves Operator and structural genes

The lac operon – An inducible operon • Bacteria are monoploid – one copy of each gene • Can make bacteria partially diploid (two copies of a gene) – Place another copy of the gene(s) on a plasmid (F’) – E. coli lac I mutant with a plasmid carrying a functional repressor gene will be designated: I - O + Z +/F’ I +

The lac operon – An inducible operon beta-galtosidase activity Genotype I+O+Z+ I - O + Z +/F’ I + O c Z +/F’ I + O + Z +/ F’ I s O c Z +/F’ I + I s O c Z -/F’ I - Lactose present + betagalactosidase activity Lactose absent -

The lac operon – An inducible operon beta-galtosidase activity Genotype Lactose present betagalactosidase activity Lactose absent I+O+Z+ + - I - O + Z +/F’ I + + - I + O c Z +/F’ I + O + Z +/ F’ I s O c Z +/F’ I + I s O c Z -/F’ I -

The lac operon – An inducible operon beta-galtosidase activity Genotype Lactose present betagalactosidase activity Lactose absent I+O+Z+ + - I - O + Z +/F’ I + + - I + O c Z +/F’ I + + + I + O + Z +/ F’ I s O c Z +/F’ I + I s O c Z -/F’ I -

plasmid E. Coli genome

The lac operon – An inducible operon beta-galtosidase activity Genotype Lactose present betagalactosidase activity Lactose absent I+O+Z+ + - I - O + Z +/F’ I + + - I + O c Z +/F’ I + + + I + O + Z +/ F’ I s - - I s O c Z +/F’ I + I s O c Z -/F’ I -

The lac operon – An inducible operon beta-galtosidase activity Genotype Lactose present betagalactosidase activity Lactose absent I+O+Z+ + - I - O + Z +/F’ I + + - I + O c Z +/F’ I + + + I + O + Z +/ F’ I s - - I s O c Z +/F’ I + + + I s O c Z -/F’ I -

The lac operon – An inducible operon beta-galtosidase activity Genotype Lactose present betagalactosidase activity Lactose absent I+O+Z+ + - I - O + Z +/F’ I + + - I + O c Z +/F’ I + + + I + O + Z +/ F’ I s - - I s O c Z +/F’ I + + + I s O c Z -/F’ I - - -

The lac operon – An inducible operon In F’ plasmid, an entire lac operon can be supplied. For example, I + O + Z - Y +/F’ I - O + Z + Y What are the beta-galactosidase and permease activities with and without lactose in the E. coli strain with the above genotype?

The lac operon • If E. coli cells are provided with both glucose and lactose, it preferentially metabolizes glucose rather than lactose. How do E. coli cells accomplish this? • Positive control and catabolite repression

Positive Control and Catabolite Repression New Players: Cyclic AMP (c. AMP) CAP (catabolite activator protein)

Positive Control and Catabolite Repression High glucose low c. AMP Low glucose high c. AMP

The c. AMP-CAP complex interacts with the promoter (neither can bind the promoter of lac operon by themselves) CAP = Catabolite Activator Protein

Facilitates the attachment of the RNA polymerase to the promoter – acts as an activator Must be a c. AMP-CAP complex to bind to the promoter

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-)

The c. AMP-CAP complex is not formed (low c. AMP), so CAP does not bind to the promoter (low) No transcription In addition, there is no inducer (lactose), so the active repressor is bound to the operator, and therefore the RNA polymerase cannot bind and transcribe the Lac operon genes

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+)

The c. AMP-CAP complex does not form (low c. AMP) The inducer (lactose) is present and inactivates the repressor • There is some transcription, but it is inefficient (no c. AMP-CAP complex)

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+) – Very little transcription

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+) – Very little transcription 3. Glucose absent (-), but lactose present (+)

The c. AMP-CAP complex does form (high c. AMP), facilitating the attachment of the RNA polymerase to the promoter The RNA polymerase is effectively attached to the promoter, enhancing transcription

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+) – Very little transcription 3. Glucose absent (-), but lactose present (+) – A lot of transcription

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+) – Very little transcription 3. Glucose absent (-), but lactose present (+) – A lot of transcription 4. Glucose and lactose both absent (-)

c. AMP pairs with CAP and can attach to the promoter No inducer (no lactose) Repressor stays bound to the operator No transcription

Positive Control and Catabolite Repression What would happen to the lac operon when: 1. Glucose present (+), but lactose absent (-) – No transcription 2. Glucose and lactose both present (+) – Very little transcription 3. Glucose absent (-), but lactose present (+) – A lot of transcription 4. Glucose and lactose both absent (-) – No transcription

Lac operon overview - animation http: //highered. mcgrawhill. com/sites/0072556781/student _view 0/chapter 12/animation_quiz _4. html