Figure 4

Comparison of ribosomal association between two halves of an mRNA by RDM. Polysomal fraction containing the majority of Yhb1 mRNA (A) or YGR026W mRNA (B) was collected from wild-type or rps9-Δ188 strains and mixed with RNase H and ODN complementary to the region indicated by an arrow on the schematic presentation of each mRNA. This should lead to a cleavage of the mRNA at the region complementary to the ODN and to result in two fragments: 5' fragment (depicted in black) and 3' fragment (depicted in white). Following the RNase H cleavage reaction, samples were separated on a sucrose gradient into 18 fractions and subjected to northern analysis. Hybridization for Yhb1 (A) was performed using a probe that recognizes the entire open reading frame, therefore both fragments appear in the same panel. Hybridization for YGR026W (B) was performed using probes specific either to the 5' or 3' fragments of the mRNA (upper and lower panels, respectively). Arrows to the left of each panel indicate the migration position of the cleavage product, as well as residual uncut mRNA ("full-length"). Histograms represent the quantitation results of the 5' fragment (black bars) and 3' fragment (white bars) of each mRNA.