The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage.
Bottom Line: In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3'UTR and stabilize it when intracellular iron become scarce.Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site.This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability.
Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.Show MeSH
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Mentions: Previous in vitro experiments have suggested that apo-AcnB bound to the 3′UTR of acnB mRNA (5). In these experiments, the authors used a 308 nucleotide-long sequence covering the 3′UTR of acnB without giving experimental evidence for the 3′ end of the transcript. We set up experiments to define the 3′ end of acnB by performing 3′RACE experiments, which indicated that the transcript terminated 67 nt after acnB ORF (Figure 2A, sequence and structure of acnB 3′UTR). Next, we performed experiments to identify the apo-AcnB binding site on acnB 3′UTR. Footprinting assays in which radiolabeled acnB 3′UTR was subjected to partial cleavage by RNase T1 (cleaves single-stranded Gs) and RNase TA (cleaves single-stranded As) in the presence of increasing amounts of purified AcnB3xFLAG protein were performed. We found clear protection of the structure surrounding the stem–loop located in 3′UTR (Figure 2B, lanes 4–11). These results suggested that apo-AcnB bound to the 3′UTR of acnB mRNA in the stem–loop structure immediately downstream of the ORF (red nucleotides in Figure 2A).
Affiliation: Department of Biochemistry, RNA Group, University of Sherbrooke, 3201 Jean Mignault Street, Sherbrooke, Quebec J1E 4K8, Canada.