Lecture 3

Question 1

What is found in the tryptophan biosynthetic operon?

Answer 1

5 genes expressed by the promoter (P) found in the tryptophan operon (O) 
trpE 
trpD 
trpC 
trpB 
trpA

Question 2

trpR- mutants:

Answer 2

• They have constitutive expression of the trp operon, it is expressed all the time regardless of any regulatory influence.

Question 3

Is TrpR a positive or negative regulatory protein?

Answer 3

• A negative regulator. The operon is expressed when levels of tryptophan are low OR repressed when levels of tryptophan are high

Question 4

Is TrpR an inducible or repressible mechanism?

Answer 4

• It is a repressible mechanism, because high levels of trp bring about low levels of expression

Question 5

TrpR binds to the Trp Operator, near the promoter of the operon. What feature does this promoter have?

Answer 5

It has dyad symmetry (5’-> 3’ is symmetrically the same as 3’->5’ with one exception), implying that the active regulatory protein binding the operator is a dimer.

Question 6

How many promoters does TrpR regulate?

Answer 6

It negatively regulates 3 promoters:

• AroG
• Trp
• TrpR (its self!)

Question 7

What do the promoters that TrpR regulates have in common?

Answer 7

• They each have a region of dyad symmetry
• They all overlap BUT…
• The position relative to the -10 and the -35 sequences differs.

Question 8

What is the lac operon?

Answer 8

• A promoter and operator with three structural genes: lacZ, lacY and lacA with the lacI gene.
• A tetramer

Question 9

What does lacZ do?

Answer 9

It cleaves the genes into their consituents (beta-galactosidase).

Question 10

What does lacY do?

Answer 10

It is involved in the transport of lactose into the cell (permease).

Question 11

What does lacA do?

Answer 11

We don’t know (transacetylase)!

Question 12

What does lacI do?

Answer 12

It synthesises repressor monomers that form tetramers which bind to the operator to block transcription of the operon.

Question 13

What is the lac repressor made up of?

Answer 13

A dimer of:

• DNA-binding domains
• Core domains 1 and 2
• Oligomerisation domains for interactions between the monomers.

Question 14

When lactose (a carbon source) is present:

Answer 14

The cell expresses the operon in order to utilise lactose. Lactose, the inducer, binds to the repressor complex so that it can no longer bind to the operator by pushing the DNA binding domains appart. RNApol binds at the promter site and mRNA is transcribed, then translated into the 3 proteins.

Question 15

What happens when you have a lacI- repressor mutant?

Answer 15

• A non-functional monomer is produced, so even in tetramer form it is defective.
• lacI- gene synthesises a defective repressor that does not bind to the DNA.
• The operon is constitutively expressed.
• Recessive to lacI+
• This is a negative regulatory mechanism (determined by the small MW effector molecule).

Question 16

What is a lacIs mutant?

Answer 16

• Non-inducible (super-respressed)
• Dominant over lacI+
• The repressor is present, and binding to the operator and it stays there regardless as to whether inducer is added or not
• Cannot bind the inducer

Question 17

What is a lacI-d mutant?

Answer 17

• Constitutive
• Dominant negative over lacI+ because the mutant monomers interfere with the function of the wild type monomers. One ‘bad’ subunit poisons the tetramer which cannot bind DNA, so the operon is expressed.
• They effect DNA binding.

Question 18

What function of a Lac monomer is affected in a lacI-d mutant?

Answer 18

A monomer has three binding functions:

• binding inducer - fine because not lacIs
• binding other monomers - fine because monomers interfere with other monomers
• binding DNA - presumably missing

Question 19

Operator constitutive mutant:

Answer 19

• Repressor cannot bind to the mutant operator
• Dominant over wild type and act in cis (if you add a WT copy of the operon the mutant is still expressed constitutively)
• It only effects expression of genes adjacent to it

Question 20

Where does RNA polymerase sit on the lac operon?

Answer 20

• Over the +1 to beyond -35 region of DNA, the same area covered by the repressor

Question 21

The lac operon has multiple operators. What are they?

Answer 21

01: essential for repression
02: in the coding sequence of the lacZ gene
03: further upstream

Question 22

How do you get full repression of the lac operon?

Answer 22

• binding of operator 01 and 02 or 03.
• if 03 or 02 are mutated there is a 2 - 4X increase in basal expression in the absence of inducer
  if 03 AND 02 are mutated there is a 100X increase in basal expression in the absence of inducer
• a mutation in 01 massive increase in basal expression

Question 23

What happens when the repressor binds at the lac repressor?

Answer 23

• It increases RNAP binding to the lac promoter while preventing transcription initiation,
• Allowing RNAP to be bound at the same time as LacI repressor allowing transcription to being immediately upon induction of the operon.