In vitro transcription assays and their application in drug discovery

Xiao Yang, Cong Ma

Research output: Journal article publicationJournal articleAcademic researchpeer-review

Abstract

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.
Original languageEnglish
Article numbere54256
JournalJournal of Visualized Experiments
Volume2016
Issue number115
DOIs
Publication statusPublished - 20 Sep 2016

Keywords

  • Antibacterial agents
  • Genetics
  • Issue 115
  • Molecular biology
  • NusA
  • RNA polymerase
  • Sigma factor
  • Transcription
  • Transcription factors

ASJC Scopus subject areas

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

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