Bacteriophage lambda Flashcards
Size of lambda genome
48515 bp, ds
Is the lambda DNA linear or circular?
Linear in head, gets circularised upon infection through bp via 12bp cohesive ends
Immediate early genes
N
cro
Delayed early genes
7 recombination genes (recombination during lytic infection + integration of lamb DNA into bacterial chromosome for lysogen int xis)
2 replication genes
cII
cIII
Q
Late genes
Lysis genes S-R
Head/tail genes A-J
Promoters
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
What does N do?
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)
What does Q do?
Suppresses termination at t3, allowing transcription from Pr’ to continue through the whole of the late genes
What is 6SRNA?
If Q is not present, transcription from Pr’ terminates at tr3, producing a 194 bp mRNA transcript called 6SRNA
How are cI levels kept low?
Gene starts directly with AUG, not with ribosome binding site! –> leads to inefficient translation, low level of protein
What elements constitute the immunity region?
Ol/Or (operator), cI, cro
Virulent mutations
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
Size of repressor protein monomer
27 kDa
Structure of repressor protein
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)
What happens if
- too much repressor protein
- sufficient
- too little
- Too much: impossible to induce lysis
- sufficient: ensures operators are occupied
- too little: induction
How do both cro and cI bind DNA?
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
Structure of operator
3 binding sites, each a palindrome with two half sites
Each individual N t of repressor protein contacts a half site
Are all the binding sites within the operator identical?
No, but conform with consensus sequence
Binding of repressor to operator
has 10x affinity for Or1/Ol1, binds there, then to adjacent sites cooperatively
No cooperative binding to site 3
What dictates cooperative interaction in repressor protein?
C terminus
Consequence of repressor binding to Or1/Ol1
Blocking transcription from Pl/Pr
Consequence of repressor binding to Or2
Interacts with sigma factor of RNA pol –> stabilises binding –> transcription from Prm
What is the active form of the repressor?
Octamer
What happens if repressor binds at Or3?
Blocks transcription of cI
How is the efficiency of induction increased?
Cooperative binding interactions –> increase sensitivity, effectively reduce levels of repressor protein present –> less of it needs to be eliminated to induce lysis
What does cIII do?
protects cII
What does cII do?
- 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)
Is Pre a strong promoter?
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
MW of Cro homodimer
9kDa
Function of cro
- binds with higher affinity to Or3, prevents transcription from Prm
- inhibits expression of early genes (eventually, when it reaches high enough concentration)
Where does the balance between lysogeny and lysis come from?
- whether cro or repressor protein gain occupancy of operator
Critical stage in decision between lysis/lysogeny:
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
How do host function affect decision between lysis and lysogen?
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
How and where does integration occur?
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
