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. 2006 Mar 1;20(5):624–636. doi: 10.1101/gad.1397906

Figure 3.

Figure 3.

UV cross-linking of 4-thioU and 6-thioG at position +4 of mRNA with 18S rRNA in 48S complexes. (A,B) RNase H digestion of 18S rRNA cross-linked to 32P-labeled (CAA)n-AUG-(CAA)m mRNA derivatives containing 4-thioU or 6-thioG at [+4] in 48S complexes assembled with (A) or without (B) eIF1. 18S rRNA was digested in the presence of DNA primers complementary to nucleotides 1634–1651 and 1797– 1814, as indicated, and analyzed by electrophoresis in denaturing 12% PAGE and autoradiography. 18S rRNA fragments to which 4-thioU or 6-thioG at [+4] of mRNA had cross-linked in 48S complexes are shown on the right. (C–E, lanes 1) Determination of exact sites of cross-linking of (CAA)n-AUG-(CAA)m mRNA derivatives containing 4-thioU or 6-thioG at [+4] to 18S rRNA in 48S complexes by primer extension analysis. (Lanes 2) In control reactions UV cross-linking was done with 48S complexes assembled on (CAA)n-AUG-(CAA)m mRNA derivatives containing 4-thioU or 6-thioG at [−3]. The positions of RT stop sites are indicated on the right. Lanes C, T, A, and G depict 18S rRNA sequence generated using the same primer. (F) Secondary structure of rabbit 18S rRNA. Positions of cross-linked nucleotides and primers used for RNase H digestion are shown as red and blue bars, respectively. (G) Part of helix 44 of 18S rRNA showing cross-linked nucleotides (red circles).