Figure 2.

Characterization of dimerization requirements for transcripts 397/444 and 1/444. (A) Temperature dependency was assayed by incubation of the RNA samples for 10 min at 23, 37, 50 and 65°C in buffer with 1 mM (left) or 5 mM (right) MgCl₂. The RNA was slowly cooled to room temperature and analyzed on a non-denaturing TBE gel. The dimerization efficiency was calculated by phosphor imager quantification and is illustrated in bar graphs. (B) Mg²⁺ requirement was measured in a 10 min incubation in buffer with 0, 0.1, 1, 2.5 and 5 mM MgCl₂, and samples were analyzed as described for (A). (C) Dimer melting curves. RNA dimers were formed at optimal dimerization conditions (10 min at 65°C in 5 mM MgCl₂ and slow cooling). After dimer formation, the sample was diluted 10-fold in water to avoid reassociation of melted RNA, and incubated for 15 min at 23, 37, 53, 65, 70, 75 and 82°C. The samples were analyzed on a TBE gel, phosphor imager analysis was performed and the percentage of remaining dimer was calculated. Black and gray coloring represents transcript 397/444 and 1/444, respectively.