FIGURE 2.

Determination of the EGS-targeting sites in the mRNAs. (A) A putative structure of yscN mRNA and RNase T1 susceptible sites. The structure was determined by the Mfold program (Zuker 2003). The cleavages at G sites from a previous report (Lundblad et al. 2008) are indicated for strong signals (filled circles) and weak signals (open circles). (B) Analysis of the cleavage products of 5′ end-labeled yscS mRNA using random EGS and RNase P. rEGSx is a random EGS library with a shortened 3′ end (Lundblad et al. 2008). (Filled triangles) Increasing concentrations of the random EGS mixture (10-, 50-, and 100-fold molar excess to yscS mRNA). (T1) Reaction product of partial RNase T1 digestion, (OH) alkaline ladder, (P) reaction product with E. coli RNase P holoenzyme, (con) untreated RNA sample. (C5) Reaction product with C5 protein. The same yscS mRNA samples were loaded twice on the 8% polyacrylamide/7 M urea gel at different points of time. (pSupS1) Internally labeled SupS1 precursor tRNA that was used as a substrate for confirming the activity of the RNase P fraction, (arrows) cleavage sites by RNase P. Note in the lefthand part of the figure, which has more resolution, the bottom band cleaved by RNase P has moved off the gel. (C) A putative structure of yscS mRNA and RNase T1 susceptible sites. (Circles) Cleavages within the coding sequence.