Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system
Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system
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DOI：
10.1261/rna.037747.112
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发表时间：
2013-05-01
期刊：
影响因子：
4.5
通讯作者：
Haniford, David B.
中科院分区：
文献类型：
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作者：
Ross, Joseph A.;Ellis, Michael J.;Haniford, David B.
Hfq在广泛的细菌中的转录后基因调控中起作用，通常通过促进共享部分序列互补性的mRNA和反式编码的sRNA的碱基配对。尚不清楚Hfq是否是顺式编码RNA配对所需的（即，反义RNA）与其靶mRNA。在目前的工作中，我们已经确定了大肠杆菌Hfq和Tn 10/IS 10反义系统的组件，RNA-IN和RNA-OUT之间的相互作用。我们表明，Hfq通过其近端RNA结合表面与RNA-OUT相互作用，这对于Hfq和反式编码的sRNA是典型的。相比之下，RNA-IN结合Hfq中的近端和远端RNA结合表面，对后者具有更高的亲和力，这是典型的sRNA-mRNA对中的mRNA相互作用。重要的是，干扰RNA与近端位点结合的Hfq中的氨基酸取代在体外负面影响RNA-IN：OUT配对，并抑制Hfq在体内负面调节IS 10转座的能力。我们还表明，Hfq与RNA-IN和RNA-OUT的结合改变了这两种RNA的二级结构元件，并推测这可能对Hfq如何促进RNA-IN：OUT配对很重要。基于这里提出的结果，我们认为，Hfq可能参与调节RNA配对在其他反义系统，包括系统编码的其他转座因子。
Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements.