Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping

Abstract

We describe a method for analyzing ternary transcription complexes, of RNA polymerase, DNA and nascent RNA32 chains, by agarose gel electrophoresis. When the RNA of such complexes is 32P-labelled, a simple comparison of the DNA fluorogram with an autoradiogram identifies transcriptionally active DNA molecules and restriction fragments in any mixture. Two limitations on the method are described: 1) retardation during electrophoresis of polymerase-DNA complexes relative to their conjugate bare NA fragments; 2) failure of very large ternary complexes to enter gels. The following potential applications of the method are surveyed: transcription unit (elongation) mapping, separation of RNA molecules in a mixture of transcripts, dinucleotide primer mapping and identification of preferred template conformations.