Reconstitution of factor-dependent, promoter proximal pausing in Drosophila Nuclear Extracts

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)133-152
Number of pages20
JournalMethods in Molecular Biology
Volume1276
DOIs
StatePublished - Jan 1 2015

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Drosophila
RNA
RNA Polymerase II
Oligonucleotides
Shock
Transcription Factors
Embryonic Structures
Hot Temperature
Gels
Antibodies
Genes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

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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.",
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Reconstitution of factor-dependent, promoter proximal pausing in Drosophila Nuclear Extracts. / Li, Jian; Gilmour, David Scott.

In: Methods in Molecular Biology, Vol. 1276, 01.01.2015, p. 133-152.

Research output: Contribution to journalArticle

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