FIGURE 3.

In vitro interaction of T. vaginalis U2/U6 catalytic domains. (A) ³²P-labeled U6 RNA (³²P-U6) was annealed with increasing concentrations of unlabeled U2 RNA (U2) and Mg²⁺ as indicated. Lower and higher arrows correspond to free ³²P-U6 and a U2–U6 complex, respectively. (B) Interaction of U2–U6 catalytic domains with a RNA oligonucleotide that mimics the intron branch site and evaluation of thermostability of the complexes. ³²P-labeled U6 RNA (³²P-U6) was annealed with unlabeled U2 RNA (U2) and 20 nM of the RNA oligo (RO), as indicated, in the presence of 20 mM of MgCl₂. Annealed complexes were then denatured at the indicated temperatures, and complex formation and stability was assessed by nondenaturing PAGE. Lower, middle, and higher arrows correspond to free ³²P-U6, the U2–U6 complex, and the U2–U6-RO complex, respectively.