Ribosome Fractionation in Yeast

Abstract

[Abstract] This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1). Figure 1. Ribosome profiles after sucrose gradient centrifugation. Ribosome fractionation was performed in the presence of CHX (black), in the absence of CHX (red) or in the presence of EDTA (green). CHX stabilizes polysomes. In the absence of CHX polysomes are destroyed but 80S is still present. 80S can be completely disrupted in cell extracts by treatment with EDTA.