Figure 2
RNase probing of SBL RNA. (a)-(c) RNA labeled with 32P at the 3'-end or (f) at the 5'-end. (a) and (f) 32P labeled RNA was treated with nucleases under the conditions minimizing self-cleavage reaction (20°C, 5 min) or (b) self-cleaved at 50°C for 60 min and self-cleavage product was analyzed either directly in the mixture for self-cleaving or (c) after re-purifying by electrophoresis in denaturing polyacrylamide gel. The lanes marked with a plus sign are reactions carried out at 20°C, pH 7.5, 10 mM MgCl2 with either no added cleavage reagent (Ct) or with ribonuclease V1 (V1), ribonuclease T1 (T1), ribonuclease A (A) and ribonuclease U2 (U2). The minus sign lanes are reactions carried out at 50°C, pH 3.5 with 7 M urea (sequencing markers). Final concentration of RNases, reaction and analysis conditions were as described under Material and Methods (See Additional file 8). Two concentrations of RNase V1 were used: 1 × 10-3 units/μl (1) or 5 × 10-3 units/μl (2). V1 products contain a 3'-hydroxyl rather than phosphate, which results in decreased mobility relative to the sequencing markers in the case of 5'-labeled RNA and increased mobility in the case of 3'-labeled RNA. Fe(II)-EDTA cleavage (lane Fe) was done to produce RNA-ladder. Note that hydroxyl radical cleavage creates 5'-labelled fragments that are one nucleotide shorter than the sequencing markers. The results of experiments shown in Figures 2(a)-(c), (f) and of others not shown are summarized on the proposed secondary structures both for original (d) and self-cleaved (e) SBL RNAs, respectively. Cut sites of ribonucleases T1, A and U2 are designated as triangles, arrows indicate sites of self-cleavage, and squares indicate positions cleaved by V1 nuclease. Only the RNase-sensitive sites qualitatively judged to be strongly cut under the condition stated are shown. The relative intensity of the triangles and squares corresponds to the relative intensity of electrophoretic bands. Green color indicates stacking area before self-cleavage, raspberry color indicates stacking area which appears after self-cleavage. Blue and black colors indicate P1 and P4 stems, respectively.