Chapter title |
Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts.
|
---|---|
Chapter number | 7 |
Book title |
Bacterial Transcriptional Control
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2392-2_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2391-5, 978-1-4939-2392-2
|
Authors |
Jian Li, David S Gilmour, David S. Gilmour, Li, Jian, Gilmour, David S. |
Abstract |
Genomic analyses reveal that RNA polymerase II initiates transcription but pauses shortly downstream on thousands of promoters in Drosophila and mammalian cells. Here, we describe the reconstitution of this promoter proximal pausing in nuclear extracts from Drosophila embryos. This approach is useful for dissecting the role(s) of transcription factors in promoter proximal pausing. Most of our studies employ the hsp70 heat shock gene promoter; however, this technique has successfully reconstituted RNA polymerase II pausing downstream of several other Drosophila promoters. A pulse/chase method is employed to restrict incorporation of radiolabel to the 5' portion of the RNA such that the specific activity of most transcripts are nearly identical and the intensity of radioactive RNA bands detected on gels reflects the molar ratios and quantities of each RNA product, regardless of length. The radiolabeled RNAs are isolated by hybridization to a biotinylated oligonucleotide and captured on magnetic beads. We also describe the use of antibodies to investigate mechanistic aspects of promoter proximal pausing. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 7 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 3 | 43% |
Professor | 1 | 14% |
Student > Bachelor | 1 | 14% |
Researcher | 1 | 14% |
Unknown | 1 | 14% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 3 | 43% |
Biochemistry, Genetics and Molecular Biology | 2 | 29% |
Unknown | 2 | 29% |