Bacteriophage lambda

Question 1

Size of lambda genome

Answer 1

48515 bp, ds

Question 2

Is the lambda DNA linear or circular?

Answer 2

Linear in head, gets circularised upon infection through bp via 12bp cohesive ends

Question 3

Immediate early genes

Answer 3

N

cro

Question 4

Delayed early genes

Answer 4

7 recombination genes (recombination during lytic infection + integration of lamb DNA into bacterial chromosome for lysogen int xis)

2 replication genes

cII

cIII

Q

Question 5

Late genes

Answer 5

Lysis genes S-R

Head/tail genes A-J

Question 6

Promoters

Answer 6

Pr: cro, then cII
Pl: N, then cIII
Pr': late genes, located between Q and S
Pi: int
Pre: repressor establishment, between cro and cII
Prm: for cI

Question 7

What does N do?

Answer 7

It suppresses termination at tr1 and tl by acting at nutR and nutL, allowing transcription of the delayed early genes, including cII (right) and cIII (left)

Question 8

What does Q do?

Answer 8

Suppresses termination at t3, allowing transcription from Pr’ to continue through the whole of the late genes

Question 9

What is 6SRNA?

Answer 9

If Q is not present, transcription from Pr’ terminates at tr3, producing a 194 bp mRNA transcript called 6SRNA

Question 10

How are cI levels kept low?

Answer 10

Gene starts directly with AUG, not with ribosome binding site! –> leads to inefficient translation, low level of protein

Question 11

What elements constitute the immunity region?

Answer 11

Ol/Or (operator), cI, cro

Question 12

Virulent mutations

Answer 12

Mutations in the operator region which prevent the repressor protein from binding –> incoming bacteriophage lambda inevitably proceeds to the lytic cycle (no immunity)

–> they allowed us to identify operator region as the site for repressor action

Question 13

Size of repressor protein monomer

Answer 13

27 kDa

Question 14

Structure of repressor protein

Answer 14

N-t DNA binding domain

C-t domain involved in dimerisation

Connector (40 aa) –> target for cleavage as a trigger of lytic growth (cleavage = repressor can’t contact DNA simultaneously)

Question 15

What happens if

• too much repressor protein
• sufficient
• too little

Answer 15

• Too much: impossible to induce lysis
• sufficient: ensures operators are occupied
• too little: induction

Question 16

How do both cro and cI bind DNA?

Answer 16

Helix turn helix motif:

Two short helical regions that fit into successive turns of the major groove of DNA

Helix 2: interactions with phosphate backbone, no specificity

Helix 3: recognition helix –> dictates specific contacts

Question 17

Structure of operator

Answer 17

3 binding sites, each a palindrome with two half sites

Each individual N t of repressor protein contacts a half site

Question 18

Are all the binding sites within the operator identical?

Answer 18

No, but conform with consensus sequence

Question 19

Binding of repressor to operator

Answer 19

has 10x affinity for Or1/Ol1, binds there, then to adjacent sites cooperatively

No cooperative binding to site 3

Question 20

What dictates cooperative interaction in repressor protein?

Answer 20

C terminus

Question 21

Consequence of repressor binding to Or1/Ol1

Answer 21

Blocking transcription from Pl/Pr

Question 22

Consequence of repressor binding to Or2

Answer 22

Interacts with sigma factor of RNA pol –> stabilises binding –> transcription from Prm

Question 23

What is the active form of the repressor?

Answer 23

Octamer

Question 24

What happens if repressor binds at Or3?

Answer 24

Blocks transcription of cI

Question 25

How is the efficiency of induction increased?

Answer 25

Cooperative binding interactions –> increase sensitivity, effectively reduce levels of repressor protein present –> less of it needs to be eliminated to induce lysis

Question 26

What does cIII do?

Answer 26

protects cII

Question 27

What does cII do?

Answer 27

• enables transcription from Pre, thus starting production of cI
• produces anti-sense transcript of cro
• transcription from Pi (int)
• transcription from Panti-Q (promoter located within the Q gene)

Question 28

Is Pre a strong promoter?

Answer 28

Unlike Prm it has a ribosome binding site, but has poor fit at -10 and no consensus at -35 –> transcription from this promoter is dependent on cII

Question 29

MW of Cro homodimer

Answer 29

9kDa

Question 30

Function of cro

Answer 30

• binds with higher affinity to Or3, prevents transcription from Prm
• inhibits expression of early genes (eventually, when it reaches high enough concentration)

Question 31

Where does the balance between lysogeny and lysis come from?

Answer 31

• whether cro or repressor protein gain occupancy of operator

Question 32

Critical stage in decision between lysis/lysogeny:

Answer 32

After early genes cease to be transcribed, and Pl/Pr cease to be transcribed and cII + cIII disappear, Prm is activated or fails to become active and pQ commits commits phage to lytic cycle

Question 33

How do host function affect decision between lysis and lysogen?

Answer 33

Proteases HflA and HflB, activated by growth on rich medium, degrade cII

If the cell is happily growing –> lysis, if it isn’t –> lysogeny

Also high M.O.I affects outcome of infection

Question 34

How and where does integration occur?

Answer 34

Int, IHF

attP and attB sites: site-specific recombination occurs via staggered cleavage and crosswise paring in stepwise fashion: first exchange generating a Holliday junction, second cycle releases structure

• occurs in intasome