Promoter trapping of c-jun promoter-binding transcription factors.
A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (-200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)(5)-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)(5) tail at each 3' end. The intact promoter and different length pieces with one or two (GT)(5) tails had almost the same capacity to bind with (AC)(5)-Sepharose. In solution, tailed c-jun promoter (60 nM) and competitor poly dI:dC (30 ng/microl) was incubated with crude HEK293 nuclear extract to form a large protein-promoter complex, and the complex was then trapped by (AC)(5)-Sepharose by centrifugation or on a column. Compared with a popular alternative method, called here the immobilized promoter method, the products of promoter trapping were purer. The preinitiation complex purified by promoter trapping had the expected components including RNA polymerase II, TATA-box binding protein (TBP), TFIIF subunit RAP74, and transcription factor SP1, and transcribed RNA in vitro. Thus, the promoter trapping approach provides a useful tool for the purification and investigation of transcription complexes.
Jiang
Daifeng
D
Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Moxley
Robert A
RA
Jarrett
Harry W
HW
D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000302:isolation & purification; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification
D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors