Promoter pBAD Strong

We decided to utilise the pBAD strong promoter (K206000), which was created by the British Columbia iGEM 2009 team as a mutagenized form of the naturally occurring pBAD promoter. The modelling was based upon and used references pertaining to the pBAD promoter, also known as the 'ara operon'. pBAD and pBAD strong are both arabinose-inducible promoters.

In the absence and presence of arabinose, the pBAD promoter acts as a repressor and an inducer respectively. The gene products of pBAD in Escherichia coli allow the cells to take up and catabolize L-arabinose, a five-carbon sugar. pBAD’s very basic structure is depicted below:

Figure 1: A basic structural outline of the pBAD and, adjacent, AraC protein, in the absence of arabinose. (Schleif, 2011)

The three enzymes that comprise the pBAD promoter cause the catabolism of the sugar arabinose as follows:

araA – arabinose isomerase which converts arabinose to ribulose

araB – ribulokinase which phosphorylates ribulose

araD – ribulose-5-phosphate epimerase which converts ribulose-5-phosphate which can then be metabolised via the pentose phosphate pathway (Bharat Patel, http://trishul.ict.griffith.edu.au/courses/3011bbs/Lecture4.pdf)

Adjacent to the 3 pBAD structural genes, there is the AraC regulatory gene. The dimeric (the compound comprises of two structurally similar subunits called monomers which together make a dimer) AraC protein actively represses transcription as well as the synthesis of the pBAD genes; this occurs when arabinose is not present in the environment. The AraC protein binds to the half sites araO₂ and ara I₁ which creates a loop within the DNA, thus blocking the RNA polymerase from binding to the pC and pBAD promoters. This can be seen in Figure 2, where it’s possible to comprehend the position of the relative half-sites.

Figure 2: The red circles in the AraC pockets denote the inducer, L-arabinose. (Schleif, 2003)

When arabinose is present, it binds to the AraC protein and this destabilises the AraC protein binding to the araI₁-O₂ half-site looped complex, but stabilizes binding to the adjacent half-sites araI₁ and araI₂, which are upstream of the pBAD promoter. This then ‘straightens’ the DNA loop, (Carra and Schleif, 1993) allowing the activation of the transcription of pBAD (Schleif, 2011).

References:

Patel, Bharat. "Regulation of Gene Expression in Prokaryotes." Lecture 4. Griffiths University. Link to paper.

Schleif, Robert. "AraC Protein: A Love-Hate Relationship." BioEssays, Vol. 25, pg. 274-282. Published 2003. Link to paper.

Schleif, Robert. The Johns Hopkins University. Last updated August 2011. Link to paper.

Carra, John and Schleif, Robert. "Variation of half-site organization and DNA looping by AraC protein" , The EMBO Journal, Vol. 12, pg. 35-44. Published 1993. Link to paper.