L4 - The lac operon 2

Question 1

In regulation of transcription, if the regulatory molecule is an ACTIVATOR protein, non-functional mutants will…

Answer 1

NOT produce B-galactosidase even when lactose present - NON-INDUCIBLE MUTANTS

Question 2

if regulatory molecule is a repressor protein, non-functional mutants will…

Answer 2

produce B-galactosidase even when lactose present - CONSTITUTIVE MUTANTS

Question 3

what is the regulatory protein of transcription of the lac operon?

Answer 3

a repressor and is the product of the i gene (lacI), i = inducibility, not in the lac operon but near

Question 4

what is the operator?

Answer 4

site where the regulatory protein binds, specific DNA sequence in operon

Question 5

if the operator is mutated so the repressor cannot bind, when will the lac operon be transcribed?

Answer 5

all the time - constitutive, as repressor cant bind and repress transcription

Question 6

operator constitutive mutants

Answer 6

constitutive mutants in position directly next to the genes in lac operon, different to i gene mutants.

Question 7

when lactose present, what causes the repressor to stop repressing?

Answer 7

the signal molecule signals presence of lactose and binds to repressor causing it to dissociate from operator.

Question 8

if signal molecule cannot bind to the repressor, when would the lac operon be transcribed?

Answer 8

never - non inducible mutants

Question 9

In a double mutant, would the lac operon be transcribed? e.g. non-inducible (prevents signal molecule binding) AND operator constitutive (repressor cant bind to operator) mutant

Answer 9

double mutants have 2 mutations working together, for the 2 mutations stated, the lac operon would be transcribed all the time - constitutive, as repressor would never have bound to operator

Question 10

what is the signal molecule in lac operon transcription?

Answer 10

ALLOLACTOSE - isomer of lactose, produced from lactose in small amounts by B-galactosidase present before induction

Question 11

summary of regulatory mechanism

Answer 11

When lactose is ABSENT, the repressor binds to operator blocking transcription.
When lactose is PRESENT, allolactose binds to the repressor and causes it to dissociate from the operator, allowing transcription.

Question 12

what is DNase protection experiment and its use?

Answer 12

identifies where protein binds to DNA, if no proteins bound to DNA the enzyme degrades. A protein bound to section of DNA protects it from DNase, allows identification of where protein exactly binds.

Question 13

what describes the operators sequence?

Answer 13

symmetrical

Question 14

what binds to the operator sequence?

Answer 14

two lac repressor polypeptides, to block the passage of RNA polymerase

Question 15

lac repressor structure

Answer 15

four subunits (polypeptides) form the repressor protein, but ONLY 2 subunits bind to one operator. Each lac repressor polypeptide has 4 domain:

1. N-terminal DNA binding domain
2. Hinge region
3. Allolactose binding central domain
4. C-terminal oligomeric assembly domain

Question 16

what are auxiliary operators?

Answer 16

In addition to the original operator, there are 2 auxiliary operators:
O2 410 bp downstream in lacZ gene
O3 83 bp upstream
(original operator is O1)

Question 17

To which operators does the lac repressor bind and why?

Answer 17

always binds to O1 - original operator - and also binds to either O2 or O3. Binding to another operator makes the binding of lac repressor much stronger and therefore reduces transcription more.
(binding is COOPERATIVE - DNA between forms loop)

Question 18

when the repressor is bound to which pair of operators is the lacZ gene partly in the loop of DNA?

Answer 18

when bound to O1 and O2

Question 19

when the repressor is bound to which pair of operators is the promoter in the loop?

Answer 19

O1 and O3

Question 20

What is the function of the hinge region on the lac repressor?

Answer 20

the structure of the hinge region holds DNA binding domains in correct orientation to bind operator cooperatively

Question 21

what causes the repressor to dissociate in presence of lactose?

Answer 21

when lactose present, allolactose binds to lac repressor and causes conformational change. Hinge region is disfigured and subunits do not bind cooperatively to operator.