Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae

Abstract

We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation. Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand. The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation. The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract. The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell. The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined. Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min.