RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone

Abstract RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces

Structure of Bacterial Regulatory RNAs Determines Their Performance in Competition for the Chaperone Protein Hfq

Bacterial regulatory RNAs require the chaperone protein Hfq to enable their pairing to mRNAs. Recent data showed that there is a hierarchy among sRNAs in the competition for access to Hfq, which could be important for the tuning of sRNA-dependent translation regulation. Here, seven structurally different sRNAs were compared using filter-based competition assays. Moreover, chimeric sRNA constructs were designed to identify structure elements important for competition performance. The data showed that besides the 3′-terminal oligouridine sequences also the 5′-terminal structure elements of sRNAs were essential for their competition performance. When the binding of sRNAs to Hfq mutants was compared, the data showed the important role of the proximal and rim sites of Hfq for the binding of six out of seven sRNAs. However, ChiX sRNA, which was the most efficient competitor, bound Hfq in a unique way using the oppositedistal and proximalfaces of this ring-shaped protein. The data indicated that the simultaneous binding to the opposite faces of Hfq was enabled by separate adenosine-rich and uridine-rich sequences in the long, single-stranded region of ChiX. Overall, the results suggest that the individual structural composition of sRNAs serves to tune their performance to different levels resulting in a hierarchy of sRNAs in the competition for access to the Hfq protein

Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation.

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.SWH and BL are funded by the Wellcome Trust (200873/Z/16/Z). This work was also supported by the NIH (R01 GM120425 to SW, F31 GM123616 to JRJ, and R01 GM078221 to JJG). KF acknowledges funding by the LMU Mentoring program of the LMU Faculty of Biology