Figure 2.

Structure probing of the genomic 3′-leader (residues 154–1). 3′- or 5′-[³²P]-endlabeled genomic RNA was probed at room temperature in 1 x TN buffer with Pb²⁺ ions (Pb²⁺) that induce hydrolysis in flexible single-stranded regions, and with RNase T1 at 37°C in 1 x TN buffer under non-denaturing conditions (T1 native), resulting in cleavage 3′ of single-stranded G residues. Control (con.): RNA incubated in 1 x TN buffer or ddH₂O (no differences were observed between the 2 conditions) without Pb²⁺ ions or RNase T1 for 17 min at room temperature; OH⁻: alkaline ladder; T1 denat.: RNase T1 cleavage at 55°C in the presence of 4 M urea. Tandem lanes (e.g., lanes 3 and 4 in panel A, T1 native.) represent 2 incubation times, 3 and 5 min for T1 native and T1 denat., 15 and 17 min for Pb²⁺ hydrolysis lanes. (A) Probing of 3′-endlabeled RNA 154–1, analyzed by 15% denaturing PAGE. (B-D). Probing of 5′-endlabeled RNA 154–1, analyzed by 8% (panels B, D) or by 15% (panel C) denaturing PAGE. Nucleotide positions are marked at the left gel margins and colored vertical lines indicate flexible single-stranded regions. (E) Secondary structure inferred from the probing data using the numbering system and colored line code as in panels A-D. RNase T1 accessibilities are depicted by filled (denaturing conditions) and open (native conditions) triangles with different symbol sizes illustrating cleavage band intensity. Sites of Pb²⁺ hydrolysis are marked correspondingly by filled circles.