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. 2002 Feb 19;99(5):2630–2635. doi: 10.1073/pnas.052596299

Figure 2.

Figure 2

Directed hydroxyl-radical cleavage of the N. crassa mt LSU intron with EPD-Fe derivatives of wild-type and variant CYT-18 proteins lacking one or both endogenous cysteine residues. A 5′-labeled in vitro transcript containing the N. crassa mt LSU intron was complexed with the indicated EPD-Fe-conjugated CYT-18 proteins, and cleavage sites were mapped in a denaturing 6% polyacrylamide gel. OH−, alkaline hydrolysis ladder; G and A, RNA sequencing ladders; lanes 1, EPD-Fe-modified protein incubated with RNA in the absence of ascorbate to initiate hydroxyl-radical cleavage; lanes 2, cleavage reactions after the addition of ascorbate; lanes 3, cleavage reactions in the presence of a 2-fold molar excess of unmodified wild-type CYT-18 protein; lanes 4, cleavage reactions in 0.5 M KCl; lanes 5, cleavage reactions in the presence of a 5-fold molar excess of nonspecific competitor RNA (pTRSE15/NsiI; ref. 30). Regions of the mt LSU intron are shown to the left, and the cleavages sites are indicated to the right.