Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping.
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.
D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009876:Operon; D012367:RNA, Viral / Q000378:metabolism; D013604:T-Phages / Q000378:metabolism; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases