Lecture 17 Flashcards
How were some genes for the lytic and lysogenic pathway found?
Some genes for lysogeny and the lytic cycle were found as Lambda forms turbid plaques (as the lysogens are immune to further infections due to repressor proteins, leading to growth), sometimes clear plaques were observed (no lysogeny) and mutants were found in cI, cII and cIII leading to always lytic pathway if mutated.
What are the key components of the induction switch region?
The switch region has three operator sites OR3, OR2 and OR1 and two promoter regions (PRM and PR on opposite sides), RNA polymerase can only bind to one promoter region, PRM needs cI as an activator, PR needs no regulatory protein. The Operator sites overlap either one (OR1 and OR3) or both promoters (OR2), they are all 17 base pairs and similar but not identical, as such Cro and cI can differentiate them. The cI protein in 95% of cases in lysogenic cells is dimeric via C terminal interactions with the N terminal binding to DNA and each OR region can bind one dimer.
What are the two controls done by CI
The switch region has three operator sites OR3, OR2 and OR1 and two promoter regions (PRM (lysogenic) and PR (lytic) on opposite sides), RNA polymerase can only bind to one promoter region, PRM needs cI as an activator, PR needs no regulatory protein. The Operator sites overlap either one (OR1 and OR3) or both promoters (OR2), they are all 17 base pairs and similar but not identical, as such Cro and cI can differentiate them. The cI protein in 95% of cases in lysogenic cells is dimeric via C terminal interactions with the N terminal binding to DNA and each OR region can bind one dimer.
What does the switch to lytic growth involve primarily and what are the two positions and a potential cause?
The switch to lytic growth involves regulatory proteins cI and Cro operator sites on the phage DNA and their promoters and RNA polymerase. The switch has two positions cI on Cro OFF (maintenance of lysogeny) or cI off and Cro on (induction of lytic). One potential cause of the switch is radiation leading to mutations.
What is the affinity for CI binding?
CI will not often bind to just OR3 or OR1 as exclusion of the genes will occur without increase, the affinity is OR2 > OR1 > OR3, once the third site is bound to cI gene utilisation will be reduced due to RNA polymerase exclusion. Bound CI at OR1 increases affinity for OR2 due to intrinsic affinity and cooperativity, binding at OR3 is weak and has no cooperativity. This is very stable and this stateis inherited by daughter cells.
How does CI cooperativity occur?
Dimers of CI lean towards each other, this cannot occur at OR3 since CI C-terminal domains are unavailable. RNA polymerase interacts at the N terminal and is therefore not affected.
How does induction into the lytic cycle occur?
UV induction can cause the prophage to enter the lytic cycle, this can occur due to DNA damage that is sensed by RecA, resulting in it becoming a co-protease, this assists CI cleavage into two domains (non functional), therefore clearing operator sites of CI. This leads to activation of PRM, repression of excision genes lost (decreases CI), RNA polymerase binds to PR and transcribes cro, Cro now determines sequence of events. Cro dimers can bind to each OR site in absence of CI, it has a single domain and is only a negative regulator with affinity OR3 > OR2 = OR1. As no more cI expression can occur the switch is thrown, PR enables phage lytic gene expression and hence the lytic cycle, later Cro shuts off own transcription and that of early phage lytic genes (Int and Xis excise prophage from DNA).
