Figure 3. Analyses of nuclear export of SSU precursors containing rpS5-ΔC.

All experiments were performed in yeast strain pGAL-RPS5 (ToY323), in which full length rpS5 is encoded under the control of the galactose inducible GAL1 promoter. The strain was either transformed with an empty vector (Yeplac195) or vectors ToP996 and ToP1101 coding for FLAG-tagged full length rpS5 or rpS5-ΔC under the control of a constitutive promoter. (A) Steady state distribution of precursor subunits. Cells were grown overnight in selective media containing galactose, diluted in YP-galactose (YPG) and expression of pGAL-RPS5 was shut down for 2 hours in YP-glucose (YPD) medium. Total DNA (DAPI) and rRNA precursors containing ITS1-sequences between site D and A₂ (ITS1-Cy3, see Fig. S1) were detected as indicated in Materials and Methods. (B) Cell fractionation after metabolic RNA labeling. After shut down of pGAL-RPS5 expression, newly synthesized RNA was labeled with [5,6-³H] uracil for 20 minutes. Nuclear (N) and cytoplasmic (C) cellular fractions were subsequently separated, RNA was extracted, separated by gel electrophoresis and newly synthesized RNA was visualized by fluorography as described in Materials and Methods.