Part:BBa_M36101:Design
Promoter, HY5 regulated (2 HY5 binding sites)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The "Promoter, HY5 regulated (2 HY5 binding sites)" is designed to be negatively regulated by the HY5 protein. As a constitutive promoter, it should continuously be promoting the gene it precedes. When HY5 binds to the binding site it should block the RNA polymerase from binding to the promoter, thus inhibiting gene expression.
Our goal was to create a constitutive promoter that was regulated by HY5 protein. HY5 is a bZIP protein that interacts with the G-box of promoter RbcS1A (1). We took the specific G-box sequence (GTGCAC). From a picture of bZIP proteins, we are pretty confident that the 6 base sequence is long enough to constitute the entire binding site for HY5 (2). We then inserted 2 binding site sequences into the constitutive promoter sequence. We looked at several variations of binding site configurations in the constitutive promoter. This configuration has one binding site between the -35 and -10 regions, and another binding site between the -10 and 0 region. We chose to put the binding sites here with the intent that HY5 binding would inhibit the RNA polymerase from binding to the promoter and transcribing the gene, thereby making HY5 a negative regulator.
Sources:
(1) http://www.plantphysiol.org/content/146/4/1862.full
(2) http://en.wikipedia.org/wiki/File:CREB.png
Source
The base of our constitutive promoter is the J23100 promoter.
(1) Details in HY5 binding procedures and binding site: http://www.plantphysiol.org/content/146/4/1862.full
(2) Evidence for HY5 binding sequence being small and the 2 3-base regions being directly adjacent to each other: http://en.wikipedia.org/wiki/File:CREB.png