Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs

Abstract

An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate). When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands. A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA. Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases. The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site. Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times [a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C]