Figure 5. Binding of wild-type and mutant Ro proteins to Y RNA and misfolded pre-5S rRNA.

A. Effect of shortening the 3’ end of pre-5S rRNA on Ro binding. ³²P-labeled misfolded X. laevis oocyte pre-5S rRNA (left) or pre-5S rRNA lacking 8 nt of the 3′ trailer (right) was incubated without protein (lane 1) or with the indicated concentrations of Ro protein (lanes 2–9). The misfolded pre-5S rRNA contains several mutations that cause it to fold efficiently into the structure recognized by Ro (Shi et al., 1996). Protein-RNA complexes were separated from unbound RNA by native gel electrophoresis.

B. Binding of mutant Ro proteins to RNA. ³²P-labeled Xenopus Y3 RNA (left panels) or misfolded pre-5S rRNA (right panels) was incubated either without protein (lane 1) or with increasing amounts of wild-type Ro (top row) or the indicated mutant proteins (bottom four rows). Protein concentrations are given in nM above lanes 2–9. Protein-RNA complexes were separated from unbound RNA in native gels.