Figure 4.

A501U-mutant ribosomes are translationally competent. (A) BY4741 cells transformed with pDP333 carrying wild-type 25S^tag rRNA or 25S^tag-A501U rRNA were grown without Dox overnight, diluted in YPDA, grown for additional 2 h, harvested and lysed in buffer S (see Methods). Lysates were centrifuged through a 15–45% sucrose gradient. Gradients were fractionated with the continuous measurement of absorbance at 254 nm to visualize ribosomal peaks. Total RNA was extracted from individual fractions and analyzed by Northern hybridizations with the 25S^tag rRNA probe FL128. (B) Schematic workflow of the translational arrest and re-initiation assay. Cell cultures shown in (A) were grown without Dox overnight, diluted in YPDA, grown for an additional 2 h, and a portion of the culture was withdrawn for gradient analysis (left). Remaining culture was collected, washed twice, resuspended in YP medium lacking dextrose and grown for 30 min (middle). Dextrose was added back to the cultures; cells were grown for additional 30 min and harvested (right). Cells were lysed in buffer F (see Methods) and lysates were analyzed by sucrose gradients as in (A).