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Team:Heidelberg/Parts/Characterization
From 2008.igem.org
Part Characterization
lambda cI (BBa_K150004)
cI protein
Bacteriophage λ cI exists as an inactive monomer at very low concentrations (<10-9 M) but forms functional homodimers at physiological concentrations that remarkably lack a global symmetry [1]. It consists of 236 amino acids, although 237 amino acids are translated since the initiator methionine is removed by the host. Although λ cI is commonly called a repressor because of its negative regulatory functions at oL and oR, cI protein acts also as a positive regulator of gene transcription and can activate transcription of its own gene through the pRM promoter in bacteriophage λ. In genetic and biochemical studies as well as through the crystal structure it has been shown, that the carboxy-terminal domain of the cI protein contains the major sites for dimerization and oligomerization [2, 3]. The cI protein has a two-domain structure with an N-terminal portion involved in DNA binding, and a C-terminal domain that mediates dimer formation, dimer-dimer interaction, and self-cleavage. The self-cleavage reaction is triggert when the lysogenic cell suffers DNA damage and depends upon an activated form of the bacterial RecA protein [2, 4-6]. The ‘hinge region’ connecting both domains contains a conserved site that can undergo this RecA-mediated autodigestion resulting in inactivation of the repressor by separating the N-terminal from the C-terminal domain of the repressor [4, 7]. The cI protein binds symmetrically to DNA, so that each amino-terminal domain contacts a similar set of bases. The N-terminal DNA-binding domain is made up of 5 α-helices of which helix 2 and helix 3 (the helix-turn-helix motif) are involved in nucleotide sequence specific DNA recognition and binding to the major groove of DNA [8-11].
Function of cI protein in bacteriophage λ
The cI repressor in bacterophage λ is the key component of a ‘genetic switch’ that enables the phage to transition from lysogenic growth to lytic development. The cI protein binds the oL and oR operator which overlap with the pL and pR promoter (the lytic promoters). This binding allows the maintenance of the lysogenic state to be governed by cI alone. As soon as cI is inactivated, e.g., by the SOS response after UV damage or other agents that cause DNA damage, the lytic development follows [12]. The two operators oL and oR both contain three binding sites for cI protein. In each case, site 1 (i.e. oL1 and oR1) has a ~10-fold greater affinity than the other sites for cI protein. The repressor, therefore, always binds first to oL1 and oR1 and than binds to the other sites in the operator in a cooperative manner [13, 14]. The carboxy-terminal domains of the repressor dimmers mediate this cooperativity which improves the specificity and strength of the cI DNA binding, enabling strong repression of the lytic promoters [15]. Furthermore, another cooperative interaction between these two sets of tetramers bound to oL and oR, 2.4 kb apart, leads to the formation of a DNA loop held by a cI octamer, i.e. two interacting tetramers, that enhances repression of the early promoters [16-19]. In this DNA-multiprotein complex, the cI dimer bound at oR2 represses pR and at the same time also stimulates pRM transcription, thus activating cI synthesis in a repressed prophage by a positive autoregulatory loop [20-22]. As the cI concentration increases because of pRM activation, two additional cI dimers are recruited to bind oL3 and oR3 to further stabilize the oL – oR loop. In this context, cI overexpression is prevented by the binding of cI to oR3 which represses pRM [17, 19]. This positive and negative autoregulation at pRM by cI ensures a narrow range of cI repressor level to be maintained, which is optimum for stable lysogeny but is at the same time adjusted low enough for efficient induction of the lysogen. By repressing transcription from the pR promoter not only expression of genes in that operon is inhibited but also phage DNA replication by preventing transcriptional activation of λ ori, the site where phage DNA replication is initiated [23]. Even if O and P functions are present this inhibition occurs and appears to be critical for establishing a lysogen [24, 25]. The stable lysogen produces sufficient repressor not only to block prophage lytic development but also to block lytic development of any extraneous infection phage, thus imparting immunity to the lysogen against lytic superinfection [26].
