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

• Published in: The Journal of biological chemistry Vol. 265; no. 16; pp. 8979 - 8982
• Main Authors: Woontner, M. (Indiana University, Bloomington, IN), Jaehning, J.A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.06.1990
• Subjects: Amanitins - pharmacology; ARN MENSAJERO; ARN MESSAGER; Biological and medical sciences; Cell Nucleus - metabolism; DNA-Binding Proteins; Fundamental and applied biological sciences. Psychology; Fungal Proteins - pharmacology; IN VITRO TRANSCRIPTION; INITIATION FACTOR; Kinetics; Magnesium - pharmacology; MESSENGER RNA; Molecular and cellular biology; Molecular genetics; Phosphoproteins; Potassium - pharmacology; Recombinant Fusion Proteins - pharmacology; RNA Polymerase II - metabolism; RNA, Fungal - biosynthesis; RNA, Messenger - biosynthesis; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Templates, Genetic; Trans-Activators; Transcription Factors; Transcription, Genetic - drug effects; Transcription. Transcription factor. Splicing. Rna processing; TRANSFERASAS; TRANSFERASE; TRANSFERASES; Uridine Triphosphate - metabolism; In vitro transcription; Enzyme; Cell extract; Site of action; Fungi; Autoradiography; Messenger RNA; RNA polymerase 2; Isolation; Ascomycetes; Kinetics; Initiation; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)38797-6

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.