Lecture 2 - Operon concept Flashcards
What is lactose hydrolyzed into?
Galactose and glucose
by B-galactosidase
What is enzyme induction?
B-galatosidase only produced when its substrate, lactose is present in the culture medium
Lactose is the inducer of the enzyme
Define inducible enzyme
Enzymes expressed when an inducer is present
Define constitutive enzyme
Continous enzyme produce regardless of the presence of inducer
What is the i- and i+ gene?
i+ is the normal inducibility gene
i- is the constitutive inducibility gene
How are genes mapped?
Genetic mapping:
Interrupted mating or disrupted conjugation experiment
What is the o+ and o(c) gene?
The second inducibility gene
o+ is normal
o(c) is constitutive
What is usually done after genetic mapping?
Complementation tests:
Testing for various mutants and wild type compensate for their defects if they are crossed with each other.
What is F’ factor?
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.
What is the z gene?
B-galactosidase
mutation (z-) causes a defect in forming active enzyme
What does it mean if i+ gene acts trans-dominant to i- mutant?
A FACTOR which is SOLUBLE and can DIFFUSE in the cell to the influence genes on a DIFFERENT piece of DNA
What does it mean if o(c) gene is cis-dominant to o+ gene?
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
What is the R gene?
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
Outline the structure of Lac operon
p > i > p > o > z > y > a
i-Repressor gene is usually found close to the other genes
What is required to synthesis the repressor protein?
mRNA
What is the i(s) gene?
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
What is the i(q) gene?
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.
What is the “minor” reaction of B-galactosidase
Galactose + Glucose -> 1,6 - Allolactose + H20
Allo-lactose is a NATURAL inducer of the Lac operon
Why is the Lac promoter naturally “leaky”?
To allow some mRNA molecules to be transcribed even in the absence of lactose.
How are the specificity of small molecules studied?
Sugar analogues, which were lactose-like molecules.
What is a gratuitous inducer?
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.
How are substrate analogues made?
Replacement of the glucose ring of lactose with other chemical groups.
What are chromogenic substrates?
Substrates that release a coloured product upon hydrolysis by B-galactosidase. For enzyme assays.
What is ONPG?
A chromogenic substrate
ONPG (Ortho Nitro Phenyl B D Galactopyranoside)
ONPG + H20 -> Galactose + ONP
ONPG is SOLUBLE, yellow coloured (A420)
