Engineered Promoters for Potent Transient Overexpression
Author(s): Dan Y. Even 1 , Adi Kedmi 1 , Shani Basch-Barzilay 1 , Diana Ideses 1 , Ravid Tikotzki 1 , Hila Shir-Shapira 1 , Orit Shefi 2 , Tamar Juven-Gershon 1 *
The transcription of protein-coding genes is a pivotal process underlying proper cellular function. The accurate initiation of transcription by RNA polymerase II (Pol II) is a critical step in the regulation of gene expression, in which Pol II is recruited to the core promoter via the basal transcription machinery (for a review, see [1,2]). The core promoter is typically 80 nucleotides long, encompassing from -40 to +40 relative to the transcription start site. In the past, it was presumed that all core promoters function via a single universal mechanism, however, it is now well established that core promoters differ in both structure and function [3-13]. The core promoter consists of several functional subregions, termed core elements or motifs. Some of the known core promoter elements are the TATA box, TFIIB recognition elements (BREu and BREd), DCE, initiator (Inr), TCT, motif ten element (MTE) and DPE [14-26]. The TATA box, which is recognized and bound by the TBP subunit of the TFIID complex, is the best-known element . The Inr motif is probably the most common element, and it encompasses the transcription start site (the A nucleotide in the Inr consensus is usually designated as position "+1", whether or not the predominant site of initiation is at this nucleotide) [9,20]. Both the MTE and DPE motifs are located downstream of the Inr and serve as recognition sites for the TAF6 and TAF9 subunits of TFIID [23,25]. The MTE and DPE function in a cooperative manner with the Inr, and a precise spacing between the Inr and each of these motifs is crucial for transcriptional activity [22-25]. There is no universal core promoter composition, and different combinations of motifs confer specific functional properties to the core promoter, e .g . the ability to function in concert with specific enhancers [27-31] or regulate developmental gene regulatory networks [32,33]. Moreover, there are core promoters that lack any of the known core motifs, suggesting the existence of additional core promoter elements that remain to be discovered.
Previous studies of the MTE motif revealed that although the MTE can function independently of the TATA box and DPE motifs, its collaboration with the TATA box, as well as with the DPE, results in strong synergy . This synergy raised the idea that by combining different core promoter elements into a single core promoter, it could be possible to design unusually strong synthetic core promoters. We have previously designed synthetic core promoters, designated Super Core Promoters (SCP1 and SCP2), which contain the TATA box, Inr, MTE and DPE elements that drive high levels of transcription both in-vivo and in-vitro ; reviewed in . To date, no natural promoters that contain such a combination of core promoter elements in a single promoter have been identified.
We now constructed an improved and potent SCP, termed SCP3. Chloramphenicol acetyltransferase (CAT) and luciferase reporter assays were...