Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.     

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.     

Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor sigmaS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of sigmaS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.     

Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA

The rpoS mRNA, which encodes the master regulator σ^S of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA–mRNA annealing process.     

Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit

Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses. DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions. In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1). DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit. The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence. In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits. We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment. The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.     

综述: 大肠杆菌RNA分子伴侣Hfq与DsrA和rpoS作用机制的研究进展

RNA分子伴侣Hfq是细菌重要的转录后调节因子,它能够帮助非编码small RNA(sRNA)与目标mRNA配对．mRNA rpoS编码的稳态sigma因子σ^S是大肠杆菌中应激响应的核心调控因子．在低温下,Hfq蛋白对于sRNA DsrA介导的mRNA rpoS的翻译激活是必需的．然而,Hfq使用何种机制来促进sRNA和mRNA配对一直有两种并不互斥的模型存在：Hfq远侧和近侧两个表面同时结合sRNA和mRNA,使得两条RNA相互靠近,便于形成碱基配对;Hfq可能结合一条或者两条RNA,改变它们的二级结构或者三级结构,从而促进sRNA-mRNA配对的实现．最近的研究报道,成功在体外捕捉到了sRNA-Hfq-mRNA三元复合物,测定了AU₆A-Hfq-A₇三元复合物的晶体结构,并且在大肠杆菌体内证实了三元复合物的形成对于Hfq帮助sRNA DsrA激活mRNA rpoS的翻译是重要的．本文以sRNA DsrA和mRNA rpoS为例综述了蛋白质Hfq与RNA的结合特性,同时也讨论了sRNA-Hfq-mRNA三元复合物的存在对于研究sRNA介导的调控机制的一些启示．     

Evaluation of the dynamic structure of DsrA RNA from E. coli and its functional consequences

DsrA RNA is an 87-nucleotide regulatory non-protein-coding RNA of Escherichia coli for which two secondary structure models (I and II) have been proposed. We have compared these models by the energy calculations, which revealed that the currently accepted model II should be rejected on the basis of thermodynamics. Here we provide new results of nuclease footprinting analysis and the application of RNA technologies that have not previously been used for DsrA RNA structural studies, such as hydrolysis with RNase H, DNAzyme, hydroxyl radicals and lead. These approaches together with bioinformatics calculations provided strong arguments for a new model III. This model clearly shows that the long U-rich region between hairpins 1 and 2 is double-stranded. These findings shed new light on DsrA RNA-Hfq interactions.     

Cosmopolitanism and the global economy: notes from China's knowledge factories

In Dalian Software Park, China's centre for IT-enabled outsourcing and offshore services, knowledge workers find themselves on the ‘assembly line’ of information processing, carrying out highly routinized, de-skilled, and poorly paid work for which they are vastly overqualified. Following the recent attention to culture and personhood in studies of global capitalism, I argue that these knowledge workers are motivated by two forms of cosmopolitanism: corporate cosmopolitanism, the capacity to reconcile the supra-territorial values of ‘global’ corporate culture with local values; and nationalist cosmopolitanism, whereby individual workers see the performance of cultural openness as a way of contributing to China's national project of modernization. As well as providing a rare account of cosmopolitanism in the workplace, this article demonstrates the significance of cosmopolitanism for the global economy. The pursuit of cosmopolitanism creates a productive friction between individual projects of self-making, corporate projects of disciplining labour, as well as national projects of pursuing modernity and development.