What Have We Learned About Synthetic Promoter Construction?
The molecular components of transcriptional regulation are modular. Transcription factors have domains for specific functions such as DNA binding, dimerization, and protein–protein interactions associated with transcriptional activation and repression. Similarly, promoters are modular. They consist of combinations of cis-acting elements that are the binding sites for transcription factors. It is this promoter architecture that largely determines the expression pattern of a gene. The modular nature of promoters is supported by the observation that many cis-acting elements retain their activities when they are taken out of their native promoter context and used as building blocks in synthetic promoters. We therefore have a large collection of cis-acting elements to use in building synthetic promoters and many minimal promoters upon which to build them. This review discusses what we have learned concerning how to use these building blocks to make synthetic promoters. It has become clear that we can increase the strength of a promoter by adding increasing numbers of cis-acting elements. However, it appears that there may be a sweet spot with regard to inducibility as promoters with increasing numbers of copies of an element often show increased background expression. Spacing between elements appears important because if elements are placed too close together activity is lost, presumably due to reduced transcription factor binding due to steric hindrance. In many cases, promoters that contain combinations of cis-acting elements show better expression characteristics than promoters that contain a single type of element. This may be because multiple transcription factor binding sites in the promoter places it at the end of multiple signal transduction pathways. Finally, some cis-acting elements form functional units with other elements and are inactive on their own. In such cases, the complete unit is required for function in a synthetic promoter. Taken together, we have learned much about how to construct synthetic promoters and this knowledge will be crucial in both designing promoters to drive transgenes and also as components of defined regulatory networks in synthetic biology.
Key wordsSynthetic promoter Cis-acting elements Synthetic biology Transgene expression Plant biotechnology
I would like to thank Peter Bowerman at BASF for critically reading this manuscript and Neal Stewart for inviting me to speak on this topic at the 2014 World Forum on Biology in Atlanta Georgia. This chapter is based on the seminar given there. I would like to thank Imre Somssich for his support and sound advice on the pathogen-inducible synthetic promoter projects. I would particularly like to thank Michael Timko for allowing me to include unpublished data on the GAG fragment in this chapter. I would also like to thank Reinhard Hehl for his patience while I was moving to a new position.
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