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. Author manuscript; available in PMC: 2016 Mar 19.
Published in final edited form as: Mol Cell. 2015 Mar 19;57(6):1099–1109. doi: 10.1016/j.molcel.2015.01.035

Figure 3. mntP riboswitch responds to MnCl2 in vivo and in vitro.

Figure 3

(A) Predicted secondary structure of the mntP aptamer based on conservation and structure probing. Nucleotides in red are conserved in >97% of the yybP-ykoY family members (Meyer et al., 2011). Blue letters correspond to mutants assayed in (B-C). See also Figure S1A and Figure S2.

(B) β-galactosidase activity for strains carrying PLlacO-5′UTRmntP-lacZ translational fusions with mutations of indicated residues grown in M9 glucose medium and incubated without and with 400 μM MnCl2 for 1 h. The results are given in Miller units as the mean ± SDM of three independent samples.

(C) Reconstitution of mntP responsiveness to manganese in vitro with a purified E. coli in vitro translation system. Wild type or mutant RNA (1 μg) encompassing the mntP 5′-UTR and mntP ORF with a C-terminal SPA tag was incubated in the presence of no metal or 400 μM MnCl2 for 2 h and then subjected to western blot analysis. The levels of the mntP-SPA mRNA were determined by primer extension analysis. The proteins were detected by silver staining of the gel after the transfer of the lower molecular weight proteins.