An in vitro transcription assay for probing drug-DNA interactions at individual drug sites.
An in vitro transcription assay of drug-DNA interactions has been described and is based largely on the stable lac UV5-initiated transcription complex. This system utilizes a synchronized population of radiolabeled nascent RNA 10 nucleotides long. Reaction of this initiated transcription complex with drug and subsequent elongation of the nascent RNA by Escherichia coli RNA polymerase, reveals blockages at drug binding sites. From these blockages it is possible to obtain four features of the drug-DNA interaction: the sequence of preferred drug binding sites, the relative drug occupancy at each binding site, the drug dissociation rate at each site, and the probability of drug-induced termination of transcription at each site. The unidirectional transcription assay has been extended to a two-promoter, counter-directed system, which yields a bidirectional transcription footprint of drug sites.
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
D R
DR
Phillips
C M
CM
Cullinane
D M
DM
Crothers
D001345:Autoradiography; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D009711:Nucleotides; D004364:Pharmaceutical Preparations; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases