Lecture 5 - Regulation of gene expression 2

Question 1

What is the structure of the L-aribinose gene and operon?

Answer 1

• 3 sets of operons responsible for the uptake and catabolism of L-arabinose
• 4 transcription units: AraE, AraFGH, AraC, AraBAD
• uptake involves araE, F, G and H
• catabolism enzymes: araB (Ribulokinase), araA (isomerise), araD (epimerase)
• AraC is the regulator: positively regulates PBAD, PFGH, PE; negatively regulates PC, PBAD
• AraC and AraBAD are on the same stretch of DNA are transcribed in opposite directions

Question 2

What is involved in the catabolism of arabinose and what is the process?

Answer 2

araBAD
araB: Ribulokinase
araA: Isomerase
araD: epimerase
1) L-arabinose -> L- ribulose (araA Isomerase)
2) L-ribulose -> L-ribulose phosphate (araB Ribulokinase)
3) L-ribulose phosphate -> D-xyulose-phosphate (araD epimerase)

Question 3

What is the araC gene in the L-arabinose operon?

Answer 3

AraC is the regulator

Controls the expression of AraFGH, AraE and AraBAD

Question 4

What are the features of the Pc and PBAD promotor regions?

Answer 4

• Pc initaties transcription in the opposite direction to the PBAD promoter
• araBAD is a structural gene
• araC is a regulatory gene
• when the araI1 and araI2 DNA binding sites upstream of pBAD are occupied by AraC this induces transcription

Question 5

What are the two basic gene regulation principles of the AraC promoter?

Answer 5

• AraC protein contacts different sites on DNA in the presence or absence of arabinose
• the mechanism by which AraC responds to the presence of arabinose is the ‘light switch mechanism’ (AraC can act as a repressor and an activator)

Question 6

What is the structure of araC?

Answer 6

• N terminal arm (18aa)
• Arabinose binding point
• DNA binding site on the C terminal end
• functions as a dimer

Question 7

How does AraC act as a repressor?

Answer 7

No arabinose, AraC = repressor

• the AraC dimer contacts two half sites on the DNA - (AraI1 and AraO2)
• this generates a DNA loop
• this prevents RNA pol from accessing the 2 promoters in the looping region
• BAD is not transcribed and araC is supressed

Question 8

How does AraC act as an activator?

Answer 8

Presence of arabinose, AraC = activator
-Arabinose binds AraC to prevent it from interacting and form the DNA loop
-AraC-arabinose binds AraI1 and AraI2 and stimulates transcription from the PBAD promoter via direct interaction with RNA pol
(cAMP-CRP must also bind to AraI to activate expression)

Question 9

What is the light switch mechanism?

Answer 9

• if no arabinose is present to bind araC, the N terminal arm of araC will stretch down and attach to the DNA binding subunit, preventing DNA binding
• if arabinose is present, araC N terminal arm will not be attached to the DNA binding subunit and AraC can bind DNA

Question 10

How does the light switch mecanism enable autoregulation of araC?

Answer 10

No arabinose
Structure enables the dimer to bind to AraO2 and AraI1 forming a loop -> therefore get no transcription of araC (OFF)
Arabinose present
-Arabinose binds AraC, freeing the DNA binding sites so the structure can bind to AraI1 and AraI2 to promote transcription (on)
-in the presence of arabinose, araC can also bind to AraOIL and AraOIR, which prevents transcription of the araC regulator

Question 11

What is the structure of the galactose operon of E.coli?

Answer 11

• 2 overlapping promoters (P2 and P1),
• 2 operators (OE and OI)
• 2 repressors (galR and galS)
• gal operon is positively regulated by CRP (cAMP receptor protein)

Question 12

What is the process of GalR negative regulation?

Answer 12

GalR is the main Gal operon repressor

• 1 dimer binds to OE and another to OI to form a tetramer
• this causes DNA looping, prevents RNA pol from being able to reach promoters P1 and P2 so turns off the transcription of galE, galT, galK and galM

Question 13

What is the process of cAMP receptor protein control of the gal operon?

Answer 13

-CRP-cAMP binds to the -35 site and activates transcription from PI (this inhibits transcription from P2)

P2 transcribes at basal levels simply in the presence of glucose

Question 14

What are the differences between catabolic and biosynthetic operons?

Answer 14

Catabolic Operons
-Operons involved in degrading compounds to obtain catabolites in order to build other molecules (enzymes encoded by lac, ara and gal operons)
Bio synthetic Operons
-Operons encode enzymes that synthesise compounds needed by the cell such as nucleotides, aa and vitamins

Regulation of biosynthetic operons is essentially opposite to that of Catabolic, but the principles are the same

Question 15

What type of operon is the Trp operon in e.coli?

Answer 15

a biosynthetic operon

Question 16

What are the features of the tryptophan operon in E.coli?

Answer 16

-single major promoter
-regulated by the trp repressor (Trpr)
-the repressor requires a corepressor (L-tryptophan) to be active
-the repressor binds to 1 of 3 sites in the operator
No L-tryptophan
-inactive repressor cannot bind to DNA so transcription can occur
L-tryptophan present
-repressor is active and can bind to DNA so transcription is repressed

Mediated by allosteric control (feedback loop)

Question 17

What type of control regulates the expression of the trp operon?

Answer 17

Negative control
e.g.
Low tryptophan = no repression
-repressor is in the inactive conformation (aporepressor) without L-tryptophan present and so cannot bind to the operator, => transcription is not inhibited
-this raises tryptophan levels
High tryptophan = repression
-if L-tryptophan (corepressor) binds aporepressor, becomes an active repressor
-can bind to operator and transcription is inhibited, lowering transcription levels of Trp synthesis genes

Question 18

What is the small molecule that induces the Galoperon?

Answer 18

D-galactose

Question 19

What is an aporepressor?

Answer 19

a repressor that is inactive until it combines with a corepressor

Question 20

What is a corepressor?

Answer 20

A molecule that combines with an aporepressor to produce the full repressor and activate its activity