Figure 5

Comparison of ribosomal association between two ends of an mRNA by RDM. A and B) Yhb1 mRNA was subjected to RDM analysis by addition of RNase H and ODN complementary to a region 314 nts (A) or 923 nts (B) downstream to the start codon (indicated by a lightning-symbol on the schematic presentation of Yhb1 mRNA). Cleavage by the RNase H and ODN at these positions is expected to yield a fragment containing the 5' third of the ORF (depicted in black) and a fragment containing the 3' third of the ORF (depicted in white). The RDM was performed on a fraction isolated from wild-type (panel i) or rps9-Δ188 (panel ii) strains. Unrelated polysomal fraction containing mRNAs associated with three ribosomes was added to each sample at the end of the reaction to serve as a common reference for the following separation step. Following the RNase H cleavage, samples were separated on a sucrose gradient into 18 fractions and subjected to northern blotting. The blots were first hybridized with Yhb1 probe (upper blots in each panel) and then with a probe to Rpp2A mRNA that sediments as associated with three ribosomes (lower blot in each panel). Arrows to the left of each panel indicate the migration position of the cleavage products as well as residual uncut mRNA ("full-length"). C) Quantitation results of the northern blots presented in A. Hatched bars in all panels present the signals of Rpp2A and black bars present the signal of the 5' fragment. D) Quantitation results of the northern blots presented in B. Hatched bars in all panels present the signals of Rpp2A and white bars present the signal of the 3' fragment.