Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli

Abstract

Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB^Glc in an RNase E/degradosome-dependent manner. The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress. In this study, we analyzed a series of ptsG-crp translational fusions to identify the mRNA region required for the rapid degradation of ptsG mRNA. We found that the ptsG-crp mRNA is destabilized in response to phosphosugar stress when it contains the 5′ portion of ptsG mRNA corresponding up to the first two transmembrane domains (TM1 and TM2) of IICB^Glc. The destabilization of ptsG-crp mRNA was largely eliminated by frameshift mutations in the transmembrane region. The IICB^Glc-CRP fusion proteins containing more than two transmembrane domains were localized at the membrane. The efficient destabilization of ptsG-crp mRNA was restored when TM1 and TM2 of IICB^Glc were replaced by part of the LacY transmembrane region. We conclude that the membrane-targeting property of IICB^Glc protein rather than the particular nucleotide or amino acid sequence is required for the efficient degradation of ptsG mRNA in response to metabolic stress. The stimulation of ptsG-crp mRNA degradation was completely eliminated when either the hfq or sgrS gene is inactivated. The efficient mRNA destabilization was observed in the absence of membrane localization when translation was reduced by introducing a mutation in the ribosome-binding site in the cytoplasmic ptsG-crp mRNA. Taken together, we conclude that mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization presumably by reducing second rounds of translation.

Keywords

• mRNA localization
• mRNA degradation
• protein targeting
• glucose transporter
• small RNA
• Hfq