For the following questions, consider these known facts about the role of cI protein in regulating the lysis/lysogeny decision in lambda phage:
1. Binding of cI protein to O_R1 inhibits transcription of the Cro gene. Thus, cI acts as a repressor of Cro gene transcription.
2. Interactions between cI protein bound to O_R2 and RNA polymerase bound to O_R3 (which also serves as the promoter for cI gene transcription) activate transcription of the cI gene. Thus, cI acts as an activator of cI gene transcription.
3. Transcription of the cI gene is inhibited by cI protein bound to O_R3. However, since the affinity of cI for O_R3 is weak, O_R3 is occupied by cI protein only when there is a high concentration of cI protein in the cell.
E. If there is a burst of cI gene expression, which function of cI protein will be observed first? Why?
F. What advantage might transcriptional inhibition confer when cI protein binds to O_R3? Why?
cI protein exists as a dimer in solution and when bound to DNA (see Figure 3). cI dimers bind cooperatively to adjacent operator sites, one repressor dimer binding to each site. The term "cooperativity" refers in this case to a positively cooperative interaction in which the binding of a cI dimer to the high affinity OR1 site increases the affinity of a second dimer for the weaker OR2 site. Thus although the OR1 and OR2 sites differ in their affinities for cI by ~10-fold, two cI dimers bind simultaneously when OR1 and OR2 sites are adjacent (Figure 3).
G. How might lambda phage benefit from the positively cooperative binding of cI to OR1 and OR2?
H. cI proteins have two domains – the N-terminal domain, which contacts DNA, and the C-terminal domain, which mediates dimer formation and the cooperative interaction between dimers (Figure 3). What would happen to cI and Cro gene expression if the only available cI protein was truncated before the C-terminal domain, such that only the N-terminal domain remained? Why?
Under normal circumstances, OR1 and OR2 are adjacent on the DNA, thus the C-terminal domains of a cI dimer bound to OR1 can interact with the C-terminal domains of a dimer bound to OR2 as shown schematically in Figure 3. Hochschild and Ptashne decided to ask what would happen to cooperative binding if the distance between OR1 and OR2 was increased.