Detailed map of a cis-regulatory input function

Abstract

Most genes are regulated by multiple transcription factors that bind specific sites in DNA regulatory regions. These cis-regulatory regions perform a computation: the rate of transcription is a function of the active concentrations of each of the input transcription factors. Here, we used accurate gene expression measurements from living cell cultures, bearing GFP reporters, to map in detail the input function of the classic lacZYA operon of Escherichia coli, as a function of about a hundred combinations of its two inducers, cAMP and isopropyl beta-d-thiogalactoside (IPTG). We found an unexpectedly intricate function with four plateau levels and four thresholds. This result compares well with a mathematical model of the binding of the regulatory proteins cAMP receptor protein (CRP) and LacI to the lac regulatory region. The model is also used to demonstrate that with few mutations, the same region could encode much purer AND-like or even OR-like functions. This possibility means that the wild-type region is selected to perform an elaborate computation in setting the transcription rate. The present approach can be generally used to map the input functions of other genes.

Fig. 1.

Simple mathematical model of the lac region, protein–DNA level reactions. Transcription occurs from states where RNAP is bound (large open arrows). The activity of the promoter in these states is α and β. An additional parameter γ describes transcription from other states (leakiness). The K parameters are the equilibrium dissociation constants of the various reactions.

Fig. 2.

Growth and GFP fluorescence of the lac-reporter strain for 20 different IPTG and cAMP concentrations. (a) OD at 600 nm. (b) GFP fluorescence (535 nm). (c) Promoter activity, d[GFP]/dt/OD₆₀₀ (proportional to mRNA production rate). Cell cycles = log₂(OD/OD₀).

Fig. 3.

lac cis-regulatory input function (promoter activity) as function of cAMP and IPTG concentrations measured by using the GFP-plasmid system. The promoter activity was taken as the mean over two cell cycles in midexponential phase; to help visualize the lowest plateau there are two rows that represent data at low IPTG. Note the logarithmic scales. (a) Promoter activity. (b) Smoothed surface plot of the promoter activity. (c) Smoothed contour plot of the promoter activity, with the four thresholds and the interdependency diagonal. (d) Schematic form of the input function of the lac promoter. The four plateaus are designated as follows: I, low cAMP, low IPTG; II, high cAMP, low IPTG; III, low cAMP, high IPTG; IV, high cAMP, high IPTG.

Fig. 4.

(a) Assay of chromosomally encoded β-galactosidase activity as a function of cAMP and IPTG concentrations by the ONPG colorimetric method. To help visualize the lowest plateau there are two rows that represent data at low IPTG. (b) Smoothed promoter activity as a function of lactose and cAMP concentration measured by using the GFP-plasmid system. Promoter activity was taken as the mean over two cell cycles in midexponential phase.

Fig. 5.

Input function (promoter activity) in the mathematical model of the lac promoter. (a) Input function with best-fit parameters to the GFP measurements. (b) Input function with parameters for AND-like function. (c) Input function with parameters for OR-like function.