Comparison of in vitro chromatin transcription using E. coli RNA polymerase and wheat germ RNA polymerase B

Abstract

Use of Escherichia coli RNA polymerase for in vitro transcription of chromatin results in the formation of double-stranded RNA molecules, which consist of a strand of endogenous mRNA and a complementary strand of de novo synthesized RNA. Unless the duplex structures are dissociated prior to isolation of the in vitro transcripts on sulfhydryl agarose columns, the endogenous mRNA can result in over-estimates of in vitro gene-specific transcription. Substitution of wheat germ RNA polymerase B for the bacterial enzyme overcomes this artifact. When mouse fetal liver chromatin is used as template, most of the mRNA synthesized by the plant enzyme is in a single-stranded form. More importantly, this synthesis is directed by a DNA template. Hybridization studies suggest that in vitro transcription of chromatin with wheat germ RNA polymerase B maintains some fidelity to genetic restrictions which operate in vivo.