Mapping the binding site of the GTX RNA probes by
RNase H digestion of the rRNA (A). The open bars
represent the 18S rRNA fragments after RNase H digestion, the sizes of
which are indicated within each bar. The shaded and solid segments
indicate the positions of GTX complementarity at nucleotides 701–741
and 1104–1136, respectively. The oligonucleotides (O1–O4) used to
direct digestion of rRNA by RNase H are represented as arrowheads. O1
is complementary to the 18S rRNA at nucleotides 632–652, O2 is
complementary at nucleotides 784–796, O3 is complementary at
nucleotides 1074–1093, and O4 is complementary at nucleotides
1174–1193. (B) Results of the RNase H digestions. After
cross-linking the GTX-1 or GTX-2 probe to dissociated ribosomes, the
RNAs were purified away from protein and annealed to the O1 or O2
oligonucleotides (for the GTX-1 probe) or to the O3 or O4
oligonucleotides (for the GTX-2 probe), and the rRNA at these positions
was digested with RNase H. The positions of the rRNAs were determined
by ethidium bromide staining of the agarose gels.