Figure 4.

Pb²⁺ and RNase footprinting of art200, tnpA_1–173 and an art200-tnpA_1–173 complex. (A) 5'³²P-labeled art200 (69 nM) was incubated in the absence or presence of increasing concentrations tnpA_1–173 (69, 138, 276, 460 or 1380 nM) before limited treatment with Pb²⁺. Note that each RNA was denatured and allowed to fold before mixing. Positions that were most strongly protected from Pb²⁺ cleavage in a tnpA-concentration dependent manner are indicated with a green asterisk. UT is untreated art200 RNA and G is an RNase T1 sequencing lane. (B) 5'³²P-labeled tnpA_1–173 (40 nM) was incubated with wild-type and mutant variants (LS’ and M1 – see Figure 1B) of art200 (600 nM) or folding buffer (-) before treatment with RNase A, T1 or V1. tnpA_1–173 and art200 RNA were denatured and allowed to fold independently before mixing, except for a control reaction with WT art200 where RNAs were mixed, denatured and allowed to fold together (FT; lanes 6, 11, 16 and 21). Nucleotide number is relative to the AUG start codon in tnpA. Nucleotides that were most strongly protected from single-strand specific RNase (A/T1) in the presence of art200 are indicated with a red asterisk and positions that showed an increased sensitivity to RNase V1 (double-strand specific) are indicated with a blue asterisk. (C) Structural constraints derived from footprinting were input into mFold to produce structures for art200 and tnpA_1–173 (see also Supplementary Figures S2 and S3). Residues in art200 that showed either weak (green circle) or strong (green circle plus asterisk) decreases in Pb2+ reactivity upon mixing with tnpA_1–173 are highlighted. Residues in tnpA_1–173 that showed strong (red circles) decreases in RNase A or T1, or strong increases (blue circles) in V1 reactivity upon art200 addition are highlighted. Two residues (−44 and −47) showed increased V1 sensitivity and decreased A1/T1 sensitivity (blue-red circles). Nucleotide changes present in M1 and M1’ versions of art200 and tnpA_1–173, respectively, are shown in bold.