Figure 6: In vitro SdsR–mutS mRNA interaction and in vivo effect of sdsR inactivation. | Nature Communications

Figure 6: In vitro SdsR–mutS mRNA interaction and in vivo effect of sdsR inactivation.

From: β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

Figure 6

(a,b) 5′-end-labelled SdsR RNA (~5 nM, SdsR*) treated with lead(II) (a) or RNase III (b) in the absence (lane −) or presence of mutS mRNA. 5′-end-labelled SdsR RNA was subjected to lead(II) cleavage for 1 or 2 min in the absence (lanes 4 and 5) or presence of cold mutS RNAs (lanes 6 and 7, ~1 μM final). The synthesized target RNA fragment comprises regions +1,222 bases to+1,539 bases downstream of the mutS start codon. Lane C: untreated SdsR RNA. Lane T1: RNase T1 ladder of hydrolysed denatured SdsR RNA. The position of cleaved G residues is given to the left of the gel (a) or to the right of the gel (b). Lane OH: alkaline ladder. Vertical bars indicate the SdsR region protected by mutS RNA. The red arrow denotes specific RNase III cleavage of SdsR in the presence of mutS mRNA, and the black arrows correspond to RNase III cleavage on SdsR alone. MutS RNA induces strong RNase III cleavage of the SdsR RNA at position U26 (red arrow in b). (c) Proposed RNA duplexes formed by SdsR with mutS mRNAs as derived from the probing results shown in a. Positions in the target sequences are relative to the start of transcription of each RNA. Vertical arrows denote RNase III cleavage of SdsR in complex with target RNAs in vitro. (d) Frequency of ampicillin-induced tetracycline-resistant mutants in the wild-type (wt), ΔsdsR and ΔsdsR ΔdinB strains. Bacteria were grown in LB (open bar) or LB supplemented with 1 μg ml−1 of ampicillin (filled bar); each result represents the mean±s.e.m. of at least three independent experiments.

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