Integration of transcriptional inputs at promoters of the arabinose catabolic pathway

Abstract

Background: Most modelling efforts of transcriptional networks involve estimations of in vivo concentrations of components, binding affinities and reaction rates, derived from in vitro biochemical assays. These assays are difficult and in vitro measurements may not approximate actual in vivo conditions. Alternatively, changes in transcription factor activity can be estimated by using partially specified models which estimate the "hidden functions" of transcription factor concentration changes; however, non-unique solutions are a potential problem. We have applied a synthetic biology approach to develop reporters that are capable of measuring transcription factor activity in vivo in real time. These synthetic reporters are comprised of a constitutive promoter with an operator site for the specific transcription factor immediately downstream. Thus, increasing transcription factor activity is measured as repression of expression of the transcription factor reporter. Measuring repression instead of activation avoids the complications of non-linear interactions between the transcription factor and RNA polymerase which differs at each promoter.

Results: Using these reporters, we show that a simple model is capable of determining the rules of integration for multiple transcriptional inputs at the four promoters of the arabinose catabolic pathway. Furthermore, we show that despite the complex and non-linear changes in cAMP-CRP activity in vivo during diauxic shift, the synthetic transcription factor reporters are capable of measuring real-time changes in transcription factor activity, and the simple model is capable of predicting the dynamic behaviour of the catabolic promoters.

Conclusions: Using a synthetic biology approach we show that the in vivo activity of transcription factors can be quantified without the need for measuring intracellular concentrations, binding affinities and reaction rates. Using measured transcription factor activity we show how different promoters can integrate common transcriptional inputs, resulting in distinct expression patterns. The data collected show that cAMP levels in vivo are dynamic and agree with observations showing that cAMP levels show a transient pulse during diauxic shift.

Figure 1

Sequence and Structure of Synthetic Transcription Factor Reporters. A. Predicted consensus of I1I2 operators. The predicted consensus from the four AraC regulated promoters is compared to the consensus from Gallegos et al. 1997. Micro. Rev. 61(4): 393 and from Seabold and Schlief 1998. JMB. 278: 529. B. Predicted CRP operator consensus from four promoters. C. Construct of the synthetic transcription factor reporters.

Figure 2

Gradient of expression in four arabinose regulon promoters. Graphs show expression data (CPS/OD) 44 minutes post induction. A. Measured expression of the four arabinose catabolic promoters. B. Predicted expression based on the mathematical model.

Figure 3

Activity profiles of the three synthetic transcription factor reporters. Expression (CPS/OD) was measured in a gradient of cAMP (0-1mM) and arabinose (0-0.2%) at 44 minutes post induction. Activities for synARA and synCRP were calculated using equation #2, and raw expression for synRNAP-σ⁷⁰ is shown.

Figure 4

Calculated transcription factor activity during diauxic shift. The period of glucose starvation is indicated by the peak in CRP-cAMP activity. Activity is calculated using equation 2 from measurements of synRNAP-σ⁷⁰, synCRP and synARA expression. These profiles represent mean expression from n = 7 experiments with time 0 normalized to the start of glucose exhaustion.

Figure 5

Simple mathematical model of promoter expression in arabinose regulon. The promoter, P, can be bound to AraC-arabinose (A*), CRP-cAMP (C*), or both. Transcription is theoretically possible in any of these states and is described by transcription activation rates α, β and γ respectively. Note that these activation rates include binding equilibria for transcription factor binding, and there is no cooperativity.

Figure 6

Observed and predicted kinetics of expression (CPS/OD) of four AraC regulon promoters, P_araBAD, P_araC, P_araE, P_araFGH, during diauxic shift. Predictions are calculated using TF activity profiles (Figure 4) and transcription activation rates estimated from model fits to steady state expression data (Table 2).