Probing the structure of the inhibitory RsaA-<i>mgr</i>A mRNA complex using enzymes and lead(II)-induced cleavages.

<p>(A) Reactions performed with 5′-end-labeled RsaA alone (lane 3) or in the presence of an excess of <i>mgr</i>A mRNA (lane 4, 25 nM; lane 5, 50 nM; lane 6, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to <i>mgr</i>A mRNA, respectively. The experiments were performed with RNase V1 (V1), lead(II) induced cleavages (Pb), Rnase T1 (T1), RNase T2 (T2) and the endoribonuclease III (RNase III). Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. The two regions protected by <i>mgr</i>A are shown by bars. (B) Enzymatic reactions performed on <i>mgr</i>A mRNA alone (lane 3) or bound to increasing concentrations of RsaA (lane 4, 25 nM; lane 5, 50 nM; lane 6, 250 nM). Same legend as in A. Lanes U, C, G, A: dideoxy-sequencing reactions. (C) Structure of the RsaA-<i>mgr</i>A mRNA complex. Summary of the enzymatic cleavages on RsaA, <i>mgr</i>A, and the complex. Enzymatic cleavages are given as follows: RNase T1 (black and plain arrow), RNase V1 (black and plain arrowhead), RNase T2 (arrow). Circled adenines and cytosines in <i>mgr</i>A mRNA are reactive towards dimethylsulfate modification. The RNase III cleavages led to RNA fragments, which terminated by a two-nucleotide 3′ overhang. Reactivity changes induced by the binding of RsaA are indicated as follows: black circles denote strong protection, enhancements and new RNase V1 cleavages are represented by asterisks. The mutations performed in RsaA and <i>mgr</i>A mRNA, are shown in the insert.