Affinity cleavage of E. coli PRNA by AOP-modified B. subtilis P protein. Cleavage of M1 RNA by AOP-modified P protein in the presence of B. subtilis pre-tRNAAsp. (A) Cleavages in the region from P1 through P4; J2/3, P3, and P4 are marked. Control lanes show base hydrolysis (OH), RNase T1, and RNase U2 ladders, and a control reaction in the presence of wild-type P protein. Nucleotide positions are numbered. (B) Secondary structure of M1 RNA catalytic domain drawn according to the topology of the T. maritima crystal structure (Frank and Pace 1998; Torres-Larios et al. 2005), indicating cleavage sites for AOP-labeled single-cysteine P protein variants: (red arrow) RNR motif; (black arrow) metal binding loop; and (black diamond) β-strand 3. Shaded arrows denote strong cleavage bands; open arrows denote weaker cleavage bands. Highlighted nucleotides indicate the effect of the protein component on Fe–EDTA cleavage of PRNA, including site protection from cleavage (red), enhancement of cleavage (blue), and variable effect (green) (Buck et al. 2005b).
