I̲ṉ V̲i̲ṯṟo̲ transcription with wheat germ RNA polymerase B

Use of E. 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 overestimates of in vitro gene-specific transcription. Substitution of wheat germ RNA polymerase B for the bacterial enzyme overcomes the major artifacts. 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.

In order to study in vitro properties of wheat germ RNA polymerase B, transcriptions were performed with various natural and artificial nucleic acid templates. The presence of a nucleic acid template appears to be an absolute requirement for catalysis by wheat germ RNA polymerase. Little, if any, homopolymer formation is found, suggesting that in vitro synthesis of RNA with this enzyme depends upon the sequence of its template. Wheat germ polymerase possesses little ability to use globin mRNA as template. With the exception of poly(rC), commerically available, single-stranded homoribonucleotides serve as poor templates. Native DNA templates are more extensively transcribed than denatured DNA templates; this differential activity on native and denatured templates appears to be insensitive to changes in ionic conditions.