Measurement of rRNA Synthesis and Degradation Rates by ³H-Uracil Labeling in Yeast

In order to measure the actual synthesis and degradation rates (SR, DR) for rRNA in yeast, we developed a method based on the pulse labeling and quantification of newly synthesized large rRNA molecules by a known mass of cells. The SR is calculated as the ratio of new rRNA molecules (synthesized after a short [5,6-³H]-uracil pulse) to total rRNA (a proxy of cell mass), calculated by northern blotting after hybridization with a ³²P-labeled rRNA probe. Then to measure the DR we perform a chase of the existing ³H-labeled rRNA for several hours during yeast culture growth. We have used this method in control experiments where the yeast cell volume varies as a way to check if the SR and DR are constant with the cell volume.