RNA polymerase II is the main promoter category driven expression of thousands of genes. Its transcription mechanisms have been widely investigated. It involves many transcription factors and a combinatorial array of cis-regulatory DNA elements. Two parts were well characterized for transcription initiation complexes, the Core promoter and the Co-regulators.

The core promoter defines as the minimal stretch of contiguous DNA sequence that is sufficient to direct accurate initiation of transcription by the RNA polymerase II. The co-regulators are other cis-acting DNA sequences including the proximal promoter, enhancers, silencers, and Boundary / insulator elements.

Core promoter: it extends either upstream or downstream for ~ 37bp at the transcription initiation site, which include BBE (TFIIB recognition element), the TATA box, Inr (initiator), and DPE (downstream promoter element) (see schematic map below, Jennifer 2002).

 

Co-regulators: The proximal promoter is the region in the immediate vicinity of the transcription start site (roughly from -250 to +250 nt). The binding sites for enhancers and repressors of transcription can be located many kbp from the transcription start site and act either to activate or to repress transcription.

Gentarget engineered the wild-type CMV promoter regions after aligned varieties of poly II promoters, and made a series of mutations inside upstream enhancer, Inr and DPE. We screened the re-engineered CMV promoter's efficiency by investigating the following aspects:

• The best enhancer in the upstream regulator region;
• The necessary of TATA box;
• How much DPE increase the transcription efficiency;
• The effects of spacer-length between transcription initiation site and the translation initiation site;
• The effects of Intron in downstream regulatory element;

 

Our proprietary suCMV promoter has 6 mutations in its enhancer region, 3 mutations inside the core promoter region, the optimized spacer distance between transcription and translation initiation sites, and an artificial intron at downstream of transcription site. Dependent upon cell types, this suCMV promoter demonstrated 4 to 6 folds higher translation efficiency than that in pCDNA3.1, and 2 to 3 fold higher than that in pcDNA3.3 (see Fig 3 below).

 

 This engineered super CMV promoter with the strongest transcription/translation efficiency was selected and integrated into our lentivrial vectors and Eco-PCR cloning expression vectors.

Ref:

1. Jennifer E.F. Butler1 and James T. Kadonaga. GENES & DEVELOPMENT 16:2583-2592, 2002.
2. Tamar JG, et al. Rational design of super core promoter that enhances gene expression. NATURE METHOD. V3(11):917-922, 2006.
3. Nikolov, D., H. et al. Crystal structure of a TFIIBTBP-TATA-element ternary complex. Nature 377: 119-128, 1995.
4. MICHAEL HAMPSEY MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 1998, p. 465-503