Lecture 2 - Operon concept

Question 1

What is lactose hydrolyzed into?

Answer 1

Galactose and glucose

by B-galactosidase

Question 2

What is enzyme induction?

Answer 2

B-galatosidase only produced when its substrate, lactose is present in the culture medium

Lactose is the inducer of the enzyme

Question 3

Define inducible enzyme

Answer 3

Enzymes expressed when an inducer is present

Question 4

Define constitutive enzyme

Answer 4

Continous enzyme produce regardless of the presence of inducer

Question 5

What is the i- and i+ gene?

Answer 5

i+ is the normal inducibility gene

i- is the constitutive inducibility gene

Question 6

How are genes mapped?

Answer 6

Genetic mapping:

Interrupted mating or disrupted conjugation experiment

Question 7

What is the o+ and o(c) gene?

Answer 7

The second inducibility gene

o+ is normal
o(c) is constitutive

Question 8

What is usually done after genetic mapping?

Answer 8

Complementation tests:

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

Question 9

What is F’ factor?

Answer 9

The fertility factor gene.
The recipient cell becomes diploid only for genes in common in the plasmid and chromosome, not for ALL the genes.

These are usually called micro-diploid, partial diploid or merodiploid.

Question 10

What is the z gene?

Answer 10

B-galactosidase

mutation (z-) causes a defect in forming active enzyme

Question 11

What does it mean if i+ gene acts trans-dominant to i- mutant?

Answer 11

A FACTOR which is SOLUBLE and can DIFFUSE in the cell to the influence genes on a DIFFERENT piece of DNA

Question 12

What does it mean if o(c) gene is cis-dominant to o+ gene?

Answer 12

o+ gene does NOT make a DIFFUSIBLE factor to influence genes on a DIFFERENT DNA molecule. It only influences genes on the SAME piece of DNA.
Its influence if confined to its own DNA

Question 13

What is the R gene?

Answer 13

A repressor factor which binds to the o gene (binding site). This factor has better affinity for lactose (inducer) hence its removed upon the presence of lactose.
Also called the i gene

Question 14

Outline the structure of Lac operon

Answer 14

p > i > p > o > z > y > a

i-Repressor gene is usually found close to the other genes

Question 15

What is required to synthesis the repressor protein?

Answer 15

mRNA

Question 16

What is the i(s) gene?

Answer 16

The i-Repressor gene produces an altered protein structure which cannot bind inducer, but can still bind to the operator.

AID: i(s) = supper repressor

Question 17

What is the i(q) gene?

Answer 17

The promoter of i-Repressor is enhanced. Resulting in overproduction of repressor proteins which have a normal working structure.

AID: i(q) = increased quantity of i gene activity.

Question 18

What is the “minor” reaction of B-galactosidase

Answer 18

Galactose + Glucose -> 1,6 - Allolactose + H20

Allo-lactose is a NATURAL inducer of the Lac operon

Question 19

Why is the Lac promoter naturally “leaky”?

Answer 19

To allow some mRNA molecules to be transcribed even in the absence of lactose.

Question 20

How are the specificity of small molecules studied?

Answer 20

Sugar analogues, which were lactose-like molecules.

Question 21

What is a gratuitous inducer?

Answer 21

An inducer protein which induces the production of enzyme but the enzyme cannot degrade it.

e.g. IPTG (Iso propyl B D Thio Galactopyranoside)
The substitution of S for O makes the glycosidic bond resistant to cleavage.

Question 22

How are substrate analogues made?

Answer 22

Replacement of the glucose ring of lactose with other chemical groups.

Question 23

What are chromogenic substrates?

Answer 23

Substrates that release a coloured product upon hydrolysis by B-galactosidase. For enzyme assays.

Question 24

What is ONPG?

Answer 24

A chromogenic substrate

ONPG (Ortho Nitro Phenyl B D Galactopyranoside)
ONPG + H20 -> Galactose + ONP

ONPG is SOLUBLE, yellow coloured (A420)

Question 25

What is X-gal?

Answer 25

A chromogenic substrate

X-gal (5 Bromo 4 Chloro 3 indolyl B D galactopyranoside)

X-gal is INSOLUBLE, DARK BLUE precipitate

Question 26

What is MUG?

Answer 26

A fluorogenic substrate

4 Methylumberlliferyl B D Galactopyranoside

MUG is FLUORESCENT and SOLUBLE