Part:BBa_K150004

lambda cI generator

Lambda cI generator consisting of a strong constitutive promotor (BBa_J23101), a ribosome binding site (BBa_B0032), a lambda cI coding sequence (BBa_K150001) and a double terminator (BBa_B0015). The lambda cI coding sequence was optimized for high expression levels in E. coli

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 569
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Bacteriophage lambda 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 o_L and o_R, cI protein acts also as a positive regulator of gene transcription and can activate transcription of its own gene through the p_RM 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 p_L and p_R 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 o_L and o_R 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 o_L1 and o_R1 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 o_L and o_R, 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 o_R2 represses p_R and at the same time also stimulates p_RM transcription, thus activating cI synthesis in a repressed prophage by a positive autoregulatory loop [20-22]. As the cI concentration increases because of p_RM activation, two additional cI dimers are recruited to bind o_L3 and o_R3 to further stabilize the o_L – o_R loop. In this context, cI overexpression is prevented by the binding of cI to o_R3 which represses p_RM [17, 19]. This positive and negative autoregulation at p_RM 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].

Figure 1: λ cI repressor dimer bound to an operator fragment. A ribbon representation of the three-dimensional structure of the dimer complex crystallized by Stayrook et al. [1]. The N-terminal domain interacts with the operator DNA, the C-terminal domain self-associates and forms the dimer contacts. The two domains are connected by a linker that tethers the N-terminal domain to the C-terminal domain.

Figure 2: Structure of the λ cI C-terminal domain octamer. A ribbon representation of the three-dimensional structure of the octameric complex crystallized by Bell and Lewis [27]. Each of the four carboxy-terminal domain dimers that comprise the octamer are colored differently. The amino-terminal residues of each subunit are indicated. Because the amino-terminal residue of each subunit in the octamer points to the outside of the structure, the DNA-binding domains would fan around the structure. Since the structure closes up to itself, no higher-ordered oligomers can be formed.