Chapter 5 Isolation, Characterization, and Translation of mRNA from Yeast

Abstract

Publisher Summary The chapter focuses on the preparation and analysis of messenger RNA (mRNA) from Saccharomyces cerevisiae. The isolation in undegraded form from yeast cells and polysomes are discussed. The chapter discusses the characterization of the 5′- and 3′-structures unique to mRNA, and its translation in an extract of wheat germ. In S. cerevisiae, most of the mRNA molecules contain relatively homogeneous polyriboadenylic acid (poly A) sequences of about 50 residues covalently linked to the 3′-end. These molecules are readily separated from total RNA by affinity chromatography on oligo-dT-cellulose columns. Columns are prepared and run at room temperature. The affinity chromatography on oligo-dT-cellulose separates the bulk of the poly-A-containing molecules from the total RNA and thus is highly suitable for preparing large quantities of poly-A-containing RNA. More complete purification of yeast mRNA are obtained using poly U immobilized on glass-fiber filters. There are no other methyl groups in yeast mRNA. These structures are resistant to nucleases and phosphatases and therefore can be easily isolated and analyzed. Wheat germ extract actively translates yeast mRNA.