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. 2014 Jul 17;42(14):9383–9398. doi: 10.1093/nar/gku630

Figure 6.

Figure 6.

(A), (B) and (C): Structure probing of LhrC4 and lapB mRNA interaction. (A) 5′ labeled lapB mRNA was partially digested with RNase T1 (5 min) or cleaved by lead(II) (2 min), either in the absence (−) or in the presence of 25- or 125-fold excess of LhrC4. As a control, untreated lapB mRNA was separated (C, lane 1). An RNase T1 ladder (T1) was separated in lane 2, and cleaved G residues are labeled along the left side of the gel. An alkaline ladder (OH) is shown in lane 3. The lapB sequence found to be protected from cleavage during LhrC4 presence is indicated on the right side of the gel. (B) Partial cleavage of 5′ labeled LhrC4 with lead(II) for 1 min (lanes 4–6) or RNase V1 for 2 min (lanes 7–9). LhrC4 was either digested alone (−) (lanes 4 and 7) or in the presence of 125- or 500-fold excess of non-labeled lapB mRNA. Untreated LhrC4 (C), an alkaline ladder (OH) and an RNase T1 ladder (T1) are shown in lane 1, 2 and 3, respectively. For an overview, selected nucleotides are labeled on the left side. Sequences showing structural changes upon lapB binding are marked on the right side of the gel: Stem loop A (lower and middle brackets; the 3′ side of stem A is marked by an asterisk) and the single-stranded stretch (upper bracket). (C) Labeled LhrC4 was partially cleaved with RNase T1 for 3 min (lanes 4–6) and lead(II) for 1 min (lanes 7–9) in the absence (−)(lanes 4 and 7) or presence of increasing amounts of non-labeled lapB mRNA (25-fold excess in lanes 5 and 8; 125-fold excess in lanes 6 and 9). Untreated LhrC4 (C), an RNase T1 (T1) and alkaline ladder (OH) are shown in lane 1, 2 and 3, respectively. On the left, the location of selected nucleotides is labeled. On the right, the observed change in secondary structure in the 3′ side of stem A upon lapB mRNA binding is marked (arrows: G48 and G52). (D) and (E) Gel mobility shift assays. In the LhrC sketches, mutated regions are shown in red. (D) lapB_mut_9 was mutated in the sequence interacting with LhrC4 and lost almost all of its capability to shift LhrC4. LhrC4_mut_9, the sRNA compensatory mutated in loop A, single-stranded stretch and terminator loop, was not shifted by lapB mRNA, but could partially compensate for the mutation in lapB_mut_9. An overview of the substitutions in lapB_mut9 and LhrC_mut_9 is shown in Supplementary Figure S17. Numbers in the bottom indicate the fraction of LhrC4 that was not shifted. (E) Mutation of only three sites in the lapB sequence (lapB_mut_10) nearly abolished all of the binding between the two RNAs. LhrC4_mut_10 which carried a corresponding mutation only in the terminator loop could still be shifted by lapB, but was also able to partially compensate for the mutation in lapB_mut_10. (F), (G) and (H) Deduced base pairing of lapB mRNA and loop A, the single-stranded stretch and the terminator loop of LhrC4, respectively. The lapB mRNA and LhrC4 sequences found to be bound in structure probing experiments is printed in bold. The start codon is marked in red. The UCCC motif is boxed. In figure (H) the three point mutations of mut_10 are indicated.