Promoter bashing
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In a laboratory setting, it may not be known that the promoter consists of two regions—single mutations can be made along the promoter, the promoter can be sequenced, and the levels of reporter assayed to find boundaries for each region.
Promoter bashing is a technique used in molecular biology to identify how certain regions of a DNA strand, commonly promoters, affect the transcription of downstream genes. Under normal circumstances, proteins bind to the promoter and activate or repress transcription. In a promoter bashing assay, specific point mutations or deletions are made in specific regions of the promoter and the transcription of the gene is then measured. The contribution of a region of the promoter can be observed by the level of transcription. If a mutation or deletion changes the level of transcription, then it is known that that region of the promoter may be a binding site or other regulatory element.[1][2][3]
Promoter bashing is often done with deletions from either the 5' or 3' end of the DNA strand; this assay is easier to perform based on repeated restriction digestion and gel-purifying fragments of specific sizes. It is often easiest to ligate the promoter into the reporter, generate a large amount of the reporter construct using PCR or growth in bacteria, and then perform serial restriction digests on this sample. The ability of upstream promoters can be easily assayed by removing segments from the 5' end, and the same for the 3' end of the strand for downstream promoters.[4]
Because promoters typically contain binding sequences for proteins that regulate transcription, these proteins are also essential when assessing the promoter's function. Proteins which associate with the promoter can be identified using an electrophoretic mobility shift assay (EMSA), and the effects of inclusion or exclusion of the proteins with the mutagenized promoters can be assessed in the assay. This allows the use of promoter bashing to not only discover the location on the DNA strand which affects transcription, but also the proteins which affect that strand. The effects of protein interactions with each other as well as the binding sites can also be assayed in this way; candidate proteins must instead be identified by protein/protein interaction assays instead of an EMSA.[5]
