In Vitro Transcription Assays and Their Application in Drug Discovery.
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.
School of Environmental and Life Sciences, University of Newcastle.
Xiao
X
Yang
School of Environmental and Life Sciences, University of Newcastle; Department of Applied Biology and Chemical Technology, The State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University; cong.ma@polyu.edu.hk.
Cong
C
Ma
D000900:Anti-Bacterial Agents; D020552:Comet Assay; D012321:DNA-Directed RNA Polymerases; D055808:Drug Discovery; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D014158:Transcription, Genetic
D000900:Anti-Bacterial Agents; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases
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