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
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.
As the promoter commonly contains binding sequences for proteins affecting transcription, those
proteins are also necessary when testing the effects of the promoter. 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

This is an example procedure for a promoter bashing assay, adapted from Boulin et al.:[6]

1. Clone the region of DNA thought to act as a promoter. Cloning is necessary for the
assay because it ensures that the promoter is the only factor affecting expression. This step
often involves extraction of the DNA from the organism it resides in and PCR amplification.
2. Sequence the region. DNA Sequencing is necessary to identify differences in mutated
promoters from the wild-type promoter, and to correlate those differences with differences in
gene expression. Additionally, it helps with the restriction digest of the region.
3. Digest with appropriate restriction endonucleases. The region can be digested to
remove elements which are though to not be part of the promoter. Additionally, the reporter
gene must be inserted a set distance from the promoter for most promoters. In some
methods of promoter bashing, multiple restriction digests are used to systematically remove
elements of the promotersthis method ensures that the regions of the promoter removed
do not contribute to reporter expression.
4. Mutagenize the promoter. Mutating the promoter is necessary if the method of removing
part of the promoter with restriction digestion is not used. Many mutated strands can be
generated, and the strands sequenced and the activities of the promoters assayed. This is
often necessary because one mutation cannot be guaranteed to inactivate a binding site.
Non-directed PCR-based mutagenesis can also be used; the parameters of the mutagenic
PCR reaction can be adjusted to introduce a reasonable number of mutations. However, the
random nature of PCR requires that more strands are assayed downstream of this step.
5. Ligate to reporter gene. The promoters to be assayed must be ligated to a reporter gene so
that gene expression levels can be measured. The reporter gene must be a sufficient
distance from the promoter that the promoter affects it as a wild-type promoter would affect
a gene. This can be verified with the positive control (full promoter).
6. Transform cells of interest with the various promoter:reporter constructs. The
promoter and reporter constructs must be ligated into a plasmid and transformed into cells in
which that plasmid can be expressed to measure the activity of each promoter sequence.
 Proteins which affect the promoter must also be added to those cells often those proteins
are placed on the same or different plasmid under the regulation of a constitutively active
promoter.
7. Measure reporter-gene transcription rates. The gene products are assayed and the rates
of reporter transcription are measured.
From the data received from assaying the different promoters, the effects of various parts of the
promoter can be ascertained. 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.