An upstream activating sequence from the Aspergillus nidulans gpdA gene.

The dual MAS1′-2′ promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2′ end relative to the corresponding 1′. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1′ promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1′–2′ TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.

Functional analysis of cis-elements, auxin response and early developmental profiles of the mannopine synthase bidirectional promoter.

Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.

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Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator.

Synthetic biology is an emergent field incorporating aspects of computer sciencemolecular biology-based methodologies in a systems biology context, taking naturallyoccurring cellular systems, pathways, and molecules, and selectively engineering themfor the generation of novel or beneficial synthetic behaviour. This study described theconstruction of a novel synthetic gene circuit, which utilises the inducible downstreamtranscriptional activation properties of the pheromone-response pathway in the buddingyeast Saccharomyces cerevisiae as the basis for initiation. The circuit was composedof three novel yeast expression plasmids; (1) a reporter plasmid in which the luciferasereporter gene was fused to the iron response element (IRE), and expressed under thecontrol of the pheromone-inducible FUS1 promoter, (2) a repressor plasmid whichconstitutively expressed the mammalian iron response protein (IRP), which can bind tothe IRE in the luciferase mRNA transcript, blocking translation, and (3) a de-repressorplasmid which also utilised the pheromone-inducible FUS1 promoter to express thebacterial LexA protein that represses transcription of the IRP gene, and thereby de-represses luciferase translation.Yeast cultures were propagated in media that selected for cells containing all threeplasmid components of the gene circuit. In these cells, during vegetative growthconditions, reporter gene translation is constitutively repressed by IRP until additionof pheromone. Upon pheromone-induction, the pheromone response pathway up-regulated the expression of the LexA protein which represses transcription of IRP,enabling the translation of luciferase, which is itself up-regulated by the pheromoneresponse pathway. The combination of the repressors functioned to increase the ratioof induction of the reporter gene between pheromone-induced and un-induced states.Proteins and mRNA species expressed by each plasmid were semi-quantified usingSDS-PAGE, Western blot, and RT-qPCR. Luciferase expression was measured using anin vitro whole cell luminescence assay, and the data used to define the circuit ?output?.Metabolic control analysis was used prior to building the circuit in silico, and identifiedthe transcription of IRP, as well as the IRP protein half-life as significant controlpoints for increasing the expression of luciferase in vivo. Modelling resulted inthe development of multiple variations of the circuit, incorporating strong and weakconstitutive promoters for the IRP. For the degradation rate, the IRP was fused with adegradation tag from the PEST rich C-terminal residue of the Cln2 protein, formingIRPPEST , with approximately a 10-fold reduced half-life compared to wild type. Byvarying the promoter strength and half-life of the IRP, the circuit could be tuned in termsof the amplitude and period of luciferase expression during pheromone induction.Simulated annealing and Hooke-Jeeves algorithms were used to estimate model pa-rameter values from the experimental luminescence data, refining the modelling suchthat it produced accurate time course simulation of the circuit output. While furthercharacterisation of the individual components would be advantageous, the constructionof the system represents a completed cycle of extensive modelling, experimentation,and further model refinement.

Engineering Tuneable Gene Circuits in Yeast

The interactions between different upstream activator sequences (UAS) and the downstream transcriptional elements of the TRP1 promoter were studied. We have inserted the UAS from the PGK gene into a series of TRP1 promoter deletions such that the PGK UAS is positioned at various distances upstream from or replaces the TRP1 UAS (UAST1). We show that activation of the TRP1 transcription unit I by the PGK UAS shows a marked position dependence, which is solely a function of the position of the PGK UAS relative to sequences involved in the determination of the RNA initiation sites in the TRP1 promoter. No cooperative activation is seen when both UASs are present in the promoter; the PGK UAS is dominant and is not repressed by the TRPI negative element. In addition, we show that the PGK and TRP1 UASs interact differently with TATA sequence at the TRP1 RNA initiation site. Our results suggest that these UASs are functionally distinct because they use different mechanisms for activating heterologous promoters.

Carol Midgely

Papers

Transcriptional activation by upstream activator sequences requires distinct interactions with downstream elements in the yeast TRP1 promoter.