T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA.
Nucleic Acids Res. 2012 Sep 1;40(17):8690-7. doi: 10.1093/nar/gks618. Epub 2012 Jul 2.
Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires base pairing with messenger RNA (mRNA) targets, which is facilitated by Hfq protein. Hfq is recruited to sRNAs and mRNAs through U-rich- and A-rich-binding sites, respectively, but their distance from the sRNA-mRNA complementary region varies widely among different genes. To determine whether distance and binding orientation affect Hfq's chaperone function, we engineered 'toy' RNAs containing strong Hfq-binding sites at defined distances from the complementary target site. We show that RNA annealing is fastest when the distal face of Hfq binds an A-rich sequence immediately 3' of the target. This recruitment advantage is lost when Hfq binds >20 nt away from the target, but is partially restored by secondary structure that shortens this distance. Although recruitment through Hfq's distal face accelerates RNA annealing, tight binding of six Us to Hfq's proximal face inhibits annealing. Finally, we show that ectopic A-rich motifs dramatically accelerate base pairing between DsrA sRNA and a minimal rpoS mRNA in the presence of Hfq, demonstrating that proximity and orientation predict the activity of Hfq on long RNAs.
小非编码 RNA（sRNA）通过与信使 RNA（mRNA）靶标进行碱基配对来调节细菌基因网络，该过程由 Hfq 蛋白辅助完成。Hfq 通过富含 U 和富含 A 的结合位点分别与 sRNA 和 mRNA 结合，但不同基因中 sRNA-mRNA 互补区的距离差异很大。为了确定距离和结合方向是否会影响 Hfq 的伴侣功能，我们构建了含有在特定距离处与互补靶位点结合的强 Hfq 结合位点的“玩具”RNA。研究表明，当 Hfq 的远端面结合靶标 3' 端的富含 A 序列时，RNA 退火最快。当 Hfq 与靶标距离>20nt 时，这种募集优势丧失，但通过缩短该距离的二级结构部分恢复。尽管通过 Hfq 的远端面募集可加速 RNA 退火，但 Hfq 近端面紧密结合六个 U 会抑制退火。最后，我们证明，在 Hfq 存在的情况下，外源富含 A 的模体可极大地加速 DsrA sRNA 与最小 rpoS mRNA 之间的碱基配对，表明距离和方向可预测 Hfq 对长 RNA 的活性。
