Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts

Research output: Chapter in Book/Report/Conference proceedingChapter

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)
Title of host publicationBacterial Transcriptional Control
Subtitle of host publicationMethods and Protocols
PublisherSpringer New York
Pages133-152
Number of pages20
ISBN (Electronic)9781493923922
ISBN (Print)9781493923915
DOIs
StatePublished - Feb 9 2015

Fingerprint

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

All Science Journal Classification (ASJC) codes

  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Li, J., & Gilmour, D. S. (2015). Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts. In Bacterial Transcriptional Control: Methods and Protocols (pp. 133-152). Springer New York. https://doi.org/10.1007/978-1-4939-2392-2_7
Li, Jian ; Gilmour, David Scott. / Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts. Bacterial Transcriptional Control: Methods and Protocols. Springer New York, 2015. pp. 133-152
@inbook{2ad7956bc57a4c6a9e9d800d05793fb2,
title = "Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts",
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.",
author = "Jian Li and Gilmour, {David Scott}",
year = "2015",
month = "2",
day = "9",
doi = "10.1007/978-1-4939-2392-2_7",
language = "English (US)",
isbn = "9781493923915",
pages = "133--152",
booktitle = "Bacterial Transcriptional Control",
publisher = "Springer New York",
address = "United States",

}

Li, J & Gilmour, DS 2015, Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts. in Bacterial Transcriptional Control: Methods and Protocols. Springer New York, pp. 133-152. https://doi.org/10.1007/978-1-4939-2392-2_7

Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts. / Li, Jian; Gilmour, David Scott.

Bacterial Transcriptional Control: Methods and Protocols. Springer New York, 2015. p. 133-152.

Research output: Chapter in Book/Report/Conference proceedingChapter

TY - CHAP

T1 - Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts

AU - Li, Jian

AU - Gilmour, David Scott

PY - 2015/2/9

Y1 - 2015/2/9

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84954623093&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84954623093&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-2392-2_7

DO - 10.1007/978-1-4939-2392-2_7

M3 - Chapter

C2 - 25665561

AN - SCOPUS:84954623093

SN - 9781493923915

SP - 133

EP - 152

BT - Bacterial Transcriptional Control

PB - Springer New York

ER -

Li J, Gilmour DS. Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts. In Bacterial Transcriptional Control: Methods and Protocols. Springer New York. 2015. p. 133-152 https://doi.org/10.1007/978-1-4939-2392-2_7