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  • Title: Analyses of mRNA destabilization and translational inhibition mediated by Hfq-binding small RNAs.
    Author: Morita T, Maki K, Yagi M, Aiba H.
    Journal: Methods Enzymol; 2008; 447():359-78. PubMed ID: 19161852.
    Abstract:
    A major class of bacterial small RNAs binds to an RNA chaperone Hfq and acts via imperfect base pairing to regulate the translation and stability of target mRNAs under specific physiological conditions. SgrS, an example for this class of small RNAs, is induced in response to the accumulation of glucose phosphates and downregulates the ptsG mRNA, which encodes the glucose transporter IICB(Glc) in Escherichia coli. SgrS forms a specific ribonucleoprotein complex with RNase E through Hfq. The regulatory outcomes of SgrS are the inhibition of translation and RNase E-dependent degradation of ptsG mRNA. Translational inhibition is the primary event for gene silencing. The crucial base pairs for the action of SgrS are confined to the 6-nt region overlapping the Shine-Dalgarno sequence of the target mRNA. Hfq accelerates the rate of duplex formation between SgrS and the target mRNA. Membrane localization of the target mRNA contributes to efficient SgrS action by competing with ribosome loading. Here, we describe major experimental methods and results used to study functions of Hfq-binding small RNAs in our laboratory. These are illustrated using the regulation of ptsG mRNA by SgrS is used as an example.
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