Figure 6.

Fluorescence quenching and cross-linking assays for IEP-ribozyme interactions.

A. Representative fluorescence quenching data. CP RNAs with fluorescein conjugated to their 5′ ends were titrated for quenching by 0-200 nM LtrA protein (Experimental Procedures). Data for all constructs tested are summarized in Supplementary Table 5.

B. Representative cross-linking data. CP RNAs with ³⁵S and azidophenacyl at their 5′ ends were incubated with LtrA protein. The complexes were UV-irradiated to induce cross-linking, and digested with RNase T1 to leave a single ³⁵S-labeled nucleotide attached to LtrA (Experimental Procedures). Samples were resolved on a 0.1%SDS/7% polyacrylamide gel, which was dried and analyzed by phosphorimaging. CYT-18 was substituted for LtrA as a specificity control, as was BSA for some experiments (not shown). Data for all positive cross-linking signals are shown in Suppl. Fig. 2, and a summary of all constructs assayed is in Supplementary Table 5.