Promoters are equivalent to "switches of gene" that control the on and off of gene expression. Transcription factors can regulated the promoter activity, which are protein molecules with the function of regulating gene expression, also known as trans-acting factors. Some transcription factors only bind to specific sequences in the promoters of their target genes, which are called cis-acting elements. The DNA-binding domains of the transcription factors covalently bind to the cis-acting elements, thus inhibiting or enhancing gene expression. Luciferase assay is an important tool for detecting the binding of transcription factors to specific cis-elements in the promoters of the target genes.
Hanbio's dual luciferase reporter system, which is commonly used to study the interaction between transcription factors and promoter, consists of the reporter plasmid pGL3-Basic, the internal reference plasmid pRL-TK and the transcription factor expression plasmid. The specific fragment of the target gene’s promoter is inserted into the upstream of the luciferase's coding sequence in pGL3-Basic vector as the promoter sequence of luciferase. Then the cells are co-transfected with pGL3-Basic, pRL-TK and the transcription factor expression plasmid. The specific luciferase substrates are added to lysates of cells. The luciferase activity can be measured by the detection of fluorescence intensity, so as to determine whether the transcription factor interacts with the fragment of the target gene's promoter. The Interaction sites between transcription factors and the promoters of target genes can be further identified by methods such as mutation of binding sites.
The Maps of pGL3-Basic and pRL-TK plasmids
Construction of promoter reporter plasmid
Construction of reporter plasmid for miRNAs and target genes
Single (Dual) Luciferase Assay Kit
The most common method for verifying the interaction between transcription factor and promoter is the dual luciferase validation assay.
Luciferase is a collective term for enzymes in nature that can catalyze luciferin to produce bioluminescence. The most representative luciferase are from Firefly and Renilla, named F-Luciferase and R-Luciferase, respectively.
These two types of luciferase act in different ways. First, the substrates and cofactors are different. F-Luciferase requires luciferin, O2, ATP, and Mg2+ for functioning, while R-Luciferase only requires coelenterazine and O2. Second, the color of bioluminescence is different. F-Luciferase catalyzes yellowish-green light with a emission wavelength of 560 nm, whereas R-Luciferase catalyzes blue light with a emission wavelength of 465 nm. It is often applied to the studys of verifying the activity that the promoter regulate gene transcription and interaction between the miRNA and the target genes.
Service content of transcription factor and promoter research:
Validation of promoter activity: Verify the strength and weakness of the promoter activity
Study on the interaction sites of transcription factors and promoters: verification of binding sites between transcription factors and promoters by truncating promoters or mutating potential binding sites.
Service process for Binding Validation of Transcription Factors and Promoters:
Contact Hanbio and leave your requirements. We will reply as soon as possible.
