Fig. 3.

Cleavage in vitro of the target rnpA mRNAs by rnpA-specific prEGS libraries. (A) E. faecalis rnpA mRNA. (B) B. subtilis rnpA mRNA. PAGE of cleavage products of 5′end-labeled rnpA mRNAs generated by RNase P holoenzyme and prEGS libraries. The RNase P holoenzyme was reconstituted by 10 nM M1 RNA and 100 nM C5 protein. rnpA mRNA cleavage products were separated on 8% polyacrylamide/7 M urea gels together with an alkali ladder, represented by OH, and a partial RNase T1 digest, represented by T1, of the same mRNA. The black triangles with prEGS labeled above represent increasing concentrations of prEGSs (10-, 100-, and 1,000-fold molar excess to the rnpA mRNAs) incubated with RNase P holoenzyme. Lane P represents reaction with RNase P holoenzyme only. Lane control means that the mRNA only was present. The numbers indicate the positions of cleavage relative to the rnpA translational start codon. The target mRNA sequences starting from the numbers (5′ to 3′) are best base-paired by the bottom EGS candidates (3′ to 5′).