Visualization of RNA polymerase bound to R-loop molecules improves electron microscopic analysis of in vitro...

An electron microscope method is described which allows improved analysis of in vitro transcription. Transcription complexes are fixed with glutaraldehyde, subjected to R-loop conditions which allow the nascent RNA chains to hybridize to the DNA templates, and mounted for electron microscopy by a protein-free preparation method. An RNA polymerase molecule (or parts of it) associated with only one end of the R-loop identifies the polarity of the transcript, thus determining the origin and direction of transcription. The method was evaluated using known in vitro promoters on the bacteriophage P1 genome and was used for mapping of additional promoters in their vicinity.