Promoter bashing

Under normal circumstances, proteins bind to the promoter and activate or repress 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.

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.

However, it is possible that there may not be enough data present and the assay must be re-run with a different promoter region and/or different mutations.

Promoter Bashing
Promoter bashing of a hypothetical two-region promoter. The promoter is cloned upstream of the lacZ reporter gene . Point mutations that inactivate each region are made (the red Xs) and the region is cloned onto a plasmid and inserted into E. coli cells, grown up, and has the presence of reporter measured. The binding of Protein B in this example is necessary for RNA polymerase to bind and initiate transcription .
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.