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.
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]
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.[5]
^Kamvysselis, M. (2003). Computational molecular genomics: genes, regulation, evolution. (Doctoral Dissertation). Retrieved from http://web.mit.edu/manoli/www/thesis/Intro.html
^Chalfie, M., & Kain, S. (2005) Methods of Biological Analyses, Green Fluorescent Protein: Properties, Applications, and Protocols. Wiley.
^Matsukura, S., Stellato, C., Plitt, J. R., Bickel, C., Miura, K., Georas, S. N., Casolaro, V., Schleimer, R. P. (1999). "Activation of Eotaxin Gene Transcription by NF-κB and STAT6 in Human Airway Epithelial Cells". J Immunol 163:6876-6883. PMID 10586089.
^Engstrom, E. M., Izhaki, A., Bowman, J. L. (2004). "Promoter bashing, microRNAs, and Knox genes. New insights, regulators, and targets-of-regulation in the establishment of lateral organ polarity in Arabidopsis." Plant Phisiol 135(2): 685-94. doi: 10.1104/pp.104.040394. PMID 15208415. PMC 514105.
^Guo, J. Y., Xu, J., Mao, D. Q., Fu, L. L., Gu, J. R., Zhu, J. D. (2002). "The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene". Cell Research 12:339-352. doi:10.1038/sj.cr.7290136.
Promoterbashing is a technique used in molecular biology to identify how certain regions of a DNA strand, commonly promoters, affect the transcription...
ISBN 0-87893-243-7. Eric M, Engstrom; Izhaki, Anat; Bowman, John L (July 2004). "PromoterBashing, microRNAs, and Knox Genes. New Insights, Regulators, and Targets-of-Regulation...
actually bound by transcription factor proteins; this process is known as promoterbashing. Organisms can have their cells transformed with a gene coding for...
drive the price of a certain share down, although this is rare. (see Stock Bashing) Actions designed to artificially raise the market price of listed securities...
list of professional wrestling promoters in the United States. This section lists notable professional wrestling promoters, especially those of the "Farmer"...
and beats up Meat Mountain. Afterwards, she is approached by wrestling promoter Bella "Big" Bucks (June Diane Raphael) of Motor City Wrestling who wants...
(June 2, 1908 – April 1, 1973) was an American professional wrestling promoter and professional sports franchise owner sometimes known as Jim Crockett...
(April 11, 1943 – August 1, 2019) was an American professional wrestler, promoter, and trainer. Race wrestled in the National Wrestling Alliance (NWA), the...
told AWA promoter Verne Gagne that he should hire him. According to Heenan, he moved his family to Atlanta after being told by GCW promoter Ole Anderson...
24, 1945) is an American businessman and former professional wrestling promoter. McMahon, along with his wife Linda, is a co-founder of the modern WWE...
2018. Helderman, Rosalind S.; Hamburger, Tom (December 14, 2017). "Music promoter dangled possible Putin meeting for Trump during campaign". The Washington...
as he would become known (for his ferocious attacks and untamable style—bashing his prey with overhand rights and wild left hooks) was able to fluctuate...
themselves. Similarly, invitations to English teams were made by private promoters or by individual clubs, such as the Melbourne Cricket Club.[citation needed]...
Vegas. After the Pacquiao–Morales rubber match, Bob Arum, Pacquiao's main promoter, announced that Manny had returned his signing bonus back to Golden Boy...
Memorial Day weekend, along with an additional day on June 4. Colorado-based promoter Barry Fey replaced Graham as Wozniak's business partner. The weather was...
actress and model Bill Graham Wulf Grajonca 1931-1991 German-American promoter Eve Graham Evelyn Beatson 1943- Scottish retired singer (The New Seekers)...
by Charlotte, North Carolina-based Jim Crockett Promotions (JCP), the promoter of the Mid-Atlantic territory immediately north of Georgia. Influential...
it to Gonzalez two days later. Flair quit IWA after problems with IWA promoter Víctor Quiñones. In January 2005, Flair appeared in Japan with All Japan...
industry as an agent, booker, color commentator, manager, photographer, promoter, trainer, and occasional professional wrestler. Cornette is widely considered...
is an Indian-born American retired professional wrestler and wrestling promoter better known by his ring name The Great Khali. He is best known for his...
the United States dates back to the days of regional territories. When a promoter ran opposition in even one town controlled by a National Wrestling Alliance...
Miniseries Command Z Kohlberg Pryce Web series Rick and Morty Fear Hole Promoter Voice, episode: "Fear No Mort" TBA The Perfect Couple † Tag Winbury Upcoming...
Global Information