Major role for mRNA binding and restructuring in sRNA recruitment by Hfq
Major role for mRNA binding and restructuring in sRNA recruitment by Hfq
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DOI：
10.1261/rna.2767211
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发表时间：
2011-08-01
期刊：
影响因子：
4.5
通讯作者：
Woodson, Sarah A.
中科院分区：
文献类型：
--
作者：
Soper, Toby J.;Doxzen, Kevin;Woodson, Sarah A.
细菌小RNA（sRNA）通过与靶mRNA的碱基配对来调节基因表达。许多sRNA需要Sm样RNA结合蛋白Hfq作为辅因子。DsrA sRNA和rpoS mRNA前导序列之间的充分表征的相互作用用于理解Hfq如何刺激sRNA与靶mRNA配对。DsrA退火通过破坏rpoS前导序列中的二级结构来刺激rpoS的表达，否则会阻止翻译。两种RNA以相似的亲和力结合Hfq，但与Hfq六聚体的相对面相互作用。使用阻断三种组分中两种之间相互作用的突变，我们证明Hfq与rpoS mRNA中功能关键（AAN）（4）基序的结合拯救了DsrA与超稳定rpoS突变体的结合。我们还表明，Hfq不能稳定地桥接RNA。只有当两个RNA互补时，才形成持久的三元复合物。因此，Hfq主要通过结合和重组rpoS mRNA起作用。然而，需要Hfq结合DsrA的最大退火在体外，这表明与两种RNA的瞬时相互作用有助于调节机制。
Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN)(4) motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.