Combinatorial transcriptional control of the lactose operon of Escherichia coli

Abstract

The goal of systems biology is to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents involved in a biological system and their complex web of interactions. The lac promoter of Escherichia coli offers the possibility of confronting "system-level" properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offer insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators Lac repressor (LacR) and cAMP-receptor protein (CRP). A previous in vivo study [Setty Y, Mayo AE, Surette MG, Alon U (2003) Proc Natl Acad Sci USA 100:7702-7707] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors that significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we were able to account for the combinatorial control of the lac promoter quantitatively, in terms of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP.

Fig. 1.

Dependence of Plac activity on the inducers. (a) The IPTG response for E. coli MG1655 and various ΔlacY mutants grown in minimal M9 medium with various amounts of IPTG and 0.5% glucose except for the cyan circles (0.5% glycerol). No cAMP was added to the medium except for the red triangles, which indicate that 1 mM cAMP was added. The lines are best fits to the Hill function (Eq. 1). (b) The cAMP response for E. coli MG1655 and various ΔcyaA mutants, grown in minimal M9 medium plus 0.5% glucose, 1 mM IPTG, and various amount of cAMP. The lines are best fits to the Hill function (Eq. 2).

Fig. 2.

Combinatorial control of Plac activity for strain TK310. (a) The cAMP-dependent loop parameter L is inferred from Eq. 4 by fixing the maximal fold-change f_IPTG to be the ratio of α₀ and the observed promoter activity in medium with no IPTG. α₀ is generated by the bare cAMP response α_cAMP by using the parameters in Table 2 (row 2). The result obtained (circles) is fitted to Eq. 5 by using a single fitting parameter, Ω = 10.3 ± 0.1, with C_cAMP = 320 μM (Table 2, row 2) and L₀ = 3.7 (Table 3, row 4). (b) The IPTG responses obtained at different cAMP levels (symbols) are plotted together with predictions of the CRP-assisted DNA-looping model (Eq. 3) with no adjustable parameters. We used R = 50 and K_IPTG = 12.3 μM (Table 3, row 3), with values of L taken from a and α_cAMP for α₀ (Eq. 2 with parameters values given in Table 2, row 2).