Lecture 8

Question 1

The lambda regulatory region:

2

Answer 1

• Contains PR (transcribes cro) and PL (transcribes N), which are transcribed by E.coli RNAP. They terminate at tR and tL
• cI, the repressor gene has it’s own promoter called PRM (for repressor maintenance), expressed in the lysogenic pathway.

Question 2

The right and left operators consist of…

and ____ and ____ bind proteins

Answer 2

• Three operator sites each with dyad symmetry, because each binds a dimer
• OR3, OR2 and OR1, which sit across PRM and PR.
• cI repressor protein and cro protein bind these operator sites.

Question 3

Lambda repressor monomer:

4

Answer 3

• Has two structural domains that form an interacting dimer.
• Activating region
• Dimerisation region (monomers bind forming a dimer)
• Tetramerization region (dimer binding dimer)

Question 4

Lambda cI dimers bind to an operator site cooperatively. This mean..
(2)

Answer 4

• cI first binds to OR1, then this binding enhances binding of cI to OR2.
• The repressor binds DNA via two alpha-helices in a dimer.

Question 5

What does repressor do when it binds to OR1 and OR2?

3

Answer 5

• cI acts negatively to represses PR
• cI acts positively to activates PRM, by enhancing RNAP binding and creating cI gene
• Some cI mutations prevent positive regulation (the activating region) without affecting negative regulation (DNA binding)

Question 6

cI acts to..

Answer 6

• Activate its own expression, while repressing cro expression, leading to repression of PR and PC and lysogeny.
• This is a positive feedback loop

Question 7

cI also binds at OL1 and OL2 which can complex with

Answer 7

• cI at OR1 and OR2 (and OR3) in the lysogen.

- it only binds when the affinity is high

Question 8

cro is a negative regulator that binds preferentially at OR3. It acts..

Answer 8

• negatively at PRM (overlaps OR3) repressing cI expression.

- as it increases cro binds at OR2 and OR1 (not cooperatively) repressing PR and it’s own expression

Question 9

During lysis cI and cro are..

5

Answer 9

• cI at OR2 and OR1
• PR off - no cro
• PRM on - more cI
• cI at OR3
• PRM off - regulates cl

Question 10

During lysogeny cI and cro are..

5

Answer 10

• cro at OR3
• PRM off - no cI
• PR on - more cro
• cro at OR2 and/orOR1
• PR off, but PR is on, thanks to Q, so all the late cascade genes will be expressed

Question 11

A plaque of WT lambda phage:

2

Answer 11

• means lysis

- it is turbid, so there are things (lysogens) growing in there.

Question 12

A clear plaque is generated by a..

1

Answer 12

• cI mutant

Question 13

What is involved in induction?

2

Answer 13

• Cleavage of cI by an E.coli protease (RecA).

- A lysogen going into the lytic cycle

Question 14

N protein:

2

Answer 14

• Acts as an anti-terminator of transcription to allow delayed early gene expression
• As the concentration of N protein builds up, more RNAPs will proceed to produced delayed early transcripts

Question 15

How does anti-termination work?

3

Answer 15

• RNAP binds and transcribes N the anti-termination protein
• Anti-termination protein binds RNAP and prevents it from recognising RNAP, allowing it to pass the terminator
• Anti-termination proteins act on specific terminators.

Question 16

nut sites:

2

Answer 16

• Termination is prevented by factors that act at nut, so rho cannot terminate.
• Are between promoters and terminators

Question 17

Anti-terminators can act at different locations in the transcription unit eg):
(3)

Answer 17

• pN acts at nutL to enable RNAP to pass tL
• pN acts the nutR to enable RNAP to pass tR1
• pQ acts at put to enable rNAP to pass tR

Question 18

N progressively allows transcription:

2

Answer 18

• As the concentration of N protein builds up, more delayed early gene expression occurs.
• As the concentration of Q protein builds up, more late genes will be expressed