Promoter trapping of c-jun promoter-binding transcription factors
| Autor: | Daifeng Jiang, Robert A. Moxley, Harry W. Jarrett |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Proto-Oncogene Proteins c-jun
Blotting Western RNA polymerase II Biochemistry Chromatography Affinity Analytical Chemistry Cell Line chemistry.chemical_compound Transcription (biology) Humans Promoter Regions Genetic Transcription factor biology Models Genetic Binding protein Organic Chemistry RNA Reproducibility of Results General Medicine Molecular biology chemistry Transcription preinitiation complex biology.protein Transcription factor II F DNA Transcription Factors |
| Zdroj: | Journal of chromatography. A. 1133(1-2) |
| ISSN: | 0021-9673 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect