Lecture 3

Question 1

Give an example of a catabolic operon

Answer 1

Lac operon

Question 2

Give an example of a biosynthetic operon

Answer 2

Trp operon

Question 3

What are biosynthetic operons involved in?

Answer 3

The expression of enzymes that synthesise small molecules (amino acids, nucleotides). If these are not present in the growth media the operon is switched on.

Question 4

Charles Yanofsky

Answer 4

Discovered the trp operon

Question 5

What are the 5 structural genes in the trp operon?
&
What do they encode?

Answer 5

trpA, tepB, tepC, trpD, trpE

The encode 5 enzymes in the biosynthetic pathway that leads to the production of a tryptophan amino acid

Question 6

Which gene codes the repressor for the trp operon?

Answer 6

trpR

Works in transT

Question 7

Trp operon: apo-repressor

Answer 7

Trp repressor

Question 8

Trp operon: holo-repressor

Answer 8

Trp repressor & tryptophan

Question 9

How does tryptophan control its own expression?

Answer 9

When there are high concentrations of tryptophan it binds the trp repressor to form the holo-repressor.
When tryptophan concentration is low tryptophan falls off the trp repressor.

Question 10

When are biosynthetic operons off?

Answer 10

When the molecule they help synthesise is available

Question 11

What is positive control?

Answer 11

The activator molecule binds to an operator region & transcription occurs.
Repression occurs when you inactivate the activator molecule
e.g. CAP proteins

Question 12

What is negative control?

Answer 12

The repressor molecule binds to an operator region to stop transcription.

Question 13

What is the arabinose operon an example of?

Answer 13

Dual control- protein with repressor & activation activity

Question 14

What is the order of genes in the arabinose operon?

Answer 14

C O I B A D

Question 15

C gene in the arabinose operon

Answer 15

ACTIVATOR PROTEIN

control gene

Question 16

O & I genes in the arabinose operon

Answer 16

AraO and AraI are the control sites

INITIATOR REGION

Question 17

B & A & D genes in the arabinose operon

Answer 17

STRUCTURAL GENES 
B = kinase
A = isomerase
D = epimerase 
together these convert the sugar arabinose to D-xylase-5-phosphate

Question 18

What happens in the presence of arabinose?

Answer 18

The operon needs to be switched on to provide enzymes involved in arabinose utilisation.
CAP + cAMP bind
AraC + arabinose bind
These act as an activator to promote transcription

Question 19

What happens if arabinose is absent?

Answer 19

AraC undergoes a conformational change so it can bind the O & I region. Operon loops to allow this binding and transcription is obstructed

Question 20

What are the two main methods for controlling transcription?

Answer 20

Choose which genes to start transcribing:
+ alternative sigma factors
+operons: co-ordinated control of gene groups

Deciding where to stop transcribing:
+anti-terminators
+attenuation

Question 21

Anti-terminators

Answer 21

Anti-termination sites have an anti-terminator protein so RNA polymerase will pick up the anti-terminator protein as it moves along the chromosome. This allows RNA polymerase to pass termination signals and transcribe subsequent genes.

Question 22

What is attenuation?

Answer 22

A regulatory mechanism for the Tryptophan operon and is focused on the leader sequence.

Question 23

Yanofsky & attenuation

Answer 23

Saw that bacterium with a deletion mutation in the Trp operon would produce more Trp operon transcripts. Transcripts were longer in these mutants.
The mutation was a deletion at the end of the leader region which was a terminator sequence.

Question 24

Describe the trpL leader region

Answer 24

146 bp and has 3 regions of inverted repeats

Question 25

Describe the inverted repeats in trpL

Answer 25

Only 1/3 of the inverted repeats is a functional terminator as it has a run of AAA/UUU. The other inverted repeats only result in stem loop formation.
The inverted repeat regions overlap.

If stem loop 1 and 3 form termination occurs as stem loop 3 is the only loop with the AAA/UUU

If stem loop 2 forms termination does not occur.

Question 26

Transcription/translation when high tryptophan

Answer 26

RNA polymerase is transcribing the leader region and transcribes the first part of the RNA that contributes to the formation of stem loop 1. The ribosome moves along behind the DNA polymerase.

RNA polymerase transcribes the region that corresponds to stem loop 2.

RNA polymerase completes transcribing the section that corresponds to stem loop 2. The ribosome finishes and dissociates. Stem loop 1 forms folding in on itself

Transcription stops as termination

Question 27

Transcription/translation when low tryptopha

Answer 27

The ribosome is following RNA polymerase

In stem loop 1 the ribosome needs 2 tryptophans so the ribosome stalls and does not dissociate.

The polymerase has completed transcribing stem loop 1 but it cannot fold as the ribosome is there. RNA polymerase completed transcribing stem loop 2.

Transcription continues as no termination