Structural probing and TPP binding affinity for various B. anthracis tenA RNA constructs. (A) Representative in-line probing data for RNA construct Thi1 (Fig. 3, nt 14–109) encompassing only aptamer 1. Full-length (FL) 5′-32P-labeled RNAs were examined after no reaction (NR), after partial digestion with RNase T1 (T1) or alkali (−OH), or after incubation with concentrations of TPP ranging from 100 fM to 10 μM (see Materials and Methods for additional details). Selected products of RNase T1 partial digestion (cleavage after G residues; closed arrowheads), sites of TPP-dependent changes in spontaneous RNA cleavage (open arrowheads). Sites labeled 1 through 3 were quantitated to estimate the K D for the RNA-ligand complex. (B) Plot of the normalized fraction of full-length Thi1 RNA cleaved at sites 1–3 versus the logarithm of the molar concentration (c) of TPP. (C) Summary of the K D values for various B. anthracis tenA RNA constructs determined by using in-line probing.
