FIGURE 5.

Dimethylsulphate protection of the sarcin/ricin loop region of the 25S rRNA. (A) Ribosomes (50 μg) of PSY and mak8-1 cells were either incubated with puromycin (2.5 or 5 mM) or buffer alone for 30 min (0 mM puromycin). All samples were then treated with DMS (35 mM), and rRNA was isolated for primer extension. Samples were separated through a 7 M urea/6% acrylamide gel. Deoxynucleotide sequencing of the 25S rDNA with the same primer used for extension indicates the nucleotides analyzed within domains V and VI of the 25S rRNA. (B) A portion of the secondary structure of the yeast 25S rRNA domains V and VI is shown. Nucleotides altered by methylation in the presence of puromycin are indicated in red underlined font and the adenine target of PAP is shown by blue boxed font. Nucleotides in black font indicate the separation range of the gel.