Transcriptional assay for probing molecular aspects of drug-DNA interactions

Abstract

An in vitro transcription assay of drug-DNA interactions has been developed largely on the basis of the stable lac -UV5-initiated transcription complex. This system comprises a synchronized population of radiolabeled nascent RNA ten nucleotides long. Reaction of drugs with this initiated transcription complex, followed by elongation of the nascent RNA by E. coli RNA polymerase, reveals blockages at drug 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 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 also been extended to a two-promoter, counter-directed system, which yields a bidirectional transcriptional footprint of drug sites. The transcription assay has recently been applied to in vitro eukaryotic systems in which drug-induced block-ages of RNA polymerase II reveal drug binding sites and the capacity of elongation factors to influence the effect of drugs at these sites.