L6 Regulation of Bacteriophage lambda

Question 1

Lambda life cycle

Answer 1

• 2 states (lytic or lysogenic), 3 potential pathways (lytic, lysogenic, induction)

Question 2

Lytic cycle

Answer 2

OneNote

Question 3

Lysogenic cycle

Answer 3

OneNote

Question 4

Lambda genome

Answer 4

• clustered by function
• most genes for lytic cycle
• not all genes expressed simultaneously, there’s a cycle to it
• has multiple promoters, PL and PR are the major promoters

Question 5

Lytic cycle - cascade of gene expression

Answer 5

• early, middle, late genes

- transition from one phase to the next uses different mechanisms

Question 6

Early genes

Answer 6

• transcribed by host RNA pol

- transcribes N (anti-terminator) and cro protein (DNA binding transcription regulator) from PL and PR promoters

Question 7

Delayed early genes

Answer 7

N protein allows transcripts to extent into delayed early genes including anti-terminator Q

Question 8

Late genes

Answer 8

Q protein allows it to extent into late genes, transcription initiates at PR’ promoter

Late genes expressed from a different promoter, not PL and PR. Expressed by PR’.

PR’ is a constitutive promoter so that RNA pol can bind and initiate transcription without an activator BUT the transcripts are terminated almost immediately in the absence of Q

Once Q gene has been expressed, Q becomes associated with RNA pol and the late genes can be completed transcribed

Question 9

PL

Answer 9

• strong constitutive promoter, no activator needed
• allows for transcription of N protein
• has tL terminator

Question 10

PR

Answer 10

• strong constitutive promoter, no activator needed
• allows for transcription of cro protein
• has tR1 terminator

Question 11

First early transcript

Answer 11

• will be terminated by tL and tR1

- transcript will only contain gene for N and cro protein

Question 12

How does anti-termination work?

Answer 12

Anti terminator binds to RNA pol so that as it progresses through, it no longer recognises the termination sequence and keeps on progressing
- anti termination proteins act on specific terminators

Question 13

Cro repressor protein

Answer 13

represses expression of C1 repressor to allow expression of lytic genes

• acts negatively at PRM repressing C1 expression
• binds preferentially to OR3

Question 14

N protein

Answer 14

• Specific for transcripts that contain a nut sequence
• Nut sequence allows for association of factors to act on RNA pol => rho cannot terminate transcription
• For N protein, doesn’t matter where nut sequence is as long as it occurs before the termination sequence
• N protein binds to mRNA that is transcribed from nut sequence

Question 15

PR’ promoter

Answer 15

• constitutive
• upstream of late genes
• required for synthesis of head and tail proteins and lysis

Question 16

Q protein

Answer 16

• interacts directly with RNA pol holoenzyme
• binds to DNA and influences RNA pol as it initiates transcription
• reduces pausing at termination sites

Question 17

Nut sequence

Answer 17

• contains BoxA and Box B

- Nut sequence allows for association of factors to act on RNA pol => rho cannot terminate transcription

Question 18

C1 repressor

Answer 18

Lambda C1 repressor binds to PL and PR
C1 repressor critical to ongoing association in a lysogenic stage - actively preventing the lytic cycle
- maintains its own expression
- binds preferentially to OR1 and OR2 (less affinity for OR2), represses PR including cro (negative)
- activates PRM (positive)

Question 19

PRM promoter

Answer 19

repressor maintenance promoter - transcribes the C1 repressor gene
- weak promoter, requires activator

Question 20

OR and OL

Answer 20

• right (and left) operator
• consists of 3 operator sites
• each operator has dyad symmetry, not identical
• two different regulatory proteins can bind these operator sites: C1 repressor (repressing lytic cycle) and cro protein (promoting lytic cycle)

OR1 operator overlaps -10 and -35 region of PR promoter
OR3 operator overlaps -10 and -35 region of PRM promoter

Question 21

C1 and cro

Answer 21

• bind to PL and PR as dimers
• binds at operators
• different affinities for O1, O2 and O3
  SEE ONENOTE

Question 22

Lambda repressor domains

Answer 22

SEE ONENOTE

• has two domains
• forms dimers which can interact to form a tetramer
• some C1 mutations prevent positive regulation without affecting negative regulation
• C1 dimers bind to operator site and can bind cooperatively
• C1 can also bind to OL1, OL2 and can complex with C1 at OR1 and OR2 (and OR3)