Lecture 7 - Lambda page Flashcards
Compare lambda phage and T-phage
They are similar but the tail section is relatively longer in lambda phage
Compare lambda phage and T-phage
They are similar but the tail section is relatively longer in lambda phage
What is lambda phages’ life cycle?
Lyse the bacteria (lytic) or remain dormant inside the cell (lysogenic) . Bacteria will grown normally until irradiated with UV
Outline the Lytic cycle of lambda phage
Infection > Cell lysis
Outline the lysogenic cycle of lambda phae
Infection > Phage DNA cyclizes >
Decision point 1 > Phage DNA replicates > Progeny phages > Cell lysis
Decision point 2 > Integration of phage DNA > Cell division > UV induction > Phage DNA dissociates > Progeny phage > Cell lysis
How small are binding domains?
What regulatory proteins do lambda phage encode?
Cro and lambda-repressor (cl)
Cro is a repressor
cl is both activator and repressor
Outline structure of cl protein
C terminal is 132-236 amino acid residues
N Terminus is 1-92 amino acid residues
it forms a dimer via C-terminal domain
N terminal domain contacts DNA, each binding a major groove
Outline structure of cro proteins
Forms dimers (homo-dimer) Also binds DNA as a dimer
No N-terminal domains, thus no cooperative binding.
What is the significance of cro and cl proteins?
They both regulate the switch between the two states of bacteria, lytic and lysogenic state.
cl binds to OR1 and OR2
cro binds to OR3 only
OR is back to back promoters in Lambda bacteriophage.
cro and cl are transcribed in OPPOSITE directions
Which protein causes the lysogenic cycle?
cl protein, bound to OR1 and OR2
This helps RNA polymerase bind and more cI repressor is produced. cl repressor almost continously bound to OR1 and 2
cl repress cro synthesis
cl repressor binds with high affinity preventing access of RNA polymerase to cro promoter
Which protein causes the lytic cycle?
cri protein, bound to OR3, preventing access of RNA polymerase to left hand promoter (cl).
In absence of cl repressor, RNA polymerase binds to cro promoter.
How does the lysogenic cycle transition to the lytic cycle?
cl protein must be removed from OR1 to enable cro synthesis and binding to OR3. This can be caused by UV light.
How does UV cause stimulation of cro activity?
UV activates a bacterial protein, RecA, converting RecA to a protein digesting enzyme.
RecA cuts cl repressor, so it cannot form stable dimers
What is the function of cl protein?
Lysogeny
Binds to the cro promoter (OR1), preventing transcription of cro (negative control)
Binds to OR2, an activator. This enhances RNA polymerase binding to produce more new repressor protein.
Can bind to OR1,2 and 3 but affinity is highest for OR1
What is the function of cro protein?
Initiate lysis
Binds to OR3
Cro is only a REPRESSOR. It prevents binding of RNA polymerase to cl protein promoter so no mRNA is transcribed.
Can bind to OR1,2,3 but affinity is highest for OR3
What is the sequence binding length of both cl and cro?
15-20bp
How do proteins recognise their DNA binding site?
The base pairs expose their EDGES. These are NOT the groups involved in complementary base pairing along the central axis of the DNA double helix.
What are palindromes?
2 fold symmetry.
OR1,2,3 sequences are similar but not identical. each are palindromic, specially at ends
What are the similarities between cl and cro?
Both recognition helices begin Glutamine-Serine
Glutamine at #1 and serine at #2 contact DNA bases 2 and 4 respectively.
These 2 bases are invarient in all operator sites
so cl and cro use identical amino acids to contact identical bases in the operators
Whats the difference between cl and cro?
Cro has asparagine at #5, which contacts position 3 in OR3
Cro has Lysine at #6, which contacts consecutive GG in OR3
cl has Alanine #6 which contacts T at OR1
cl and cro have the same helices orientation
What determines if phage lambda should replicate or remain dormant?
If there is plenty of food, it will replicate. Otherwise it will remain dormant.
What are the 3 stages to the phage gene expression following entry of phage DNA into bacteria?
Early
Delayed early
Late
Early and delayed early phases of gene expression occur before the decisions to switch to either lysogeny or the lytic phase.
In the late stage of gene expression, the switch operates.
Lytic cycle: early genes turned off. structural and lytic turned on.
Lysogenic: only 2 genes turned on,cl repressor and INT.
INT gene mediates the integration of the phae DNA into the bacterial chromosomes.
What is lambda phages’ life cycle?
Lyse the bacteria (lytic) or remain dormant inside the cell (lysogenic) . Bacteria will grown normally until irradiated with UV
