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Recognition of the Small Regulatory RNA Rydc by the 1 Bacterial Hfq

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Recognition of the Small Regulatory RNA Rydc by the 1 Bacterial Hfq 1 Recognition of the small regulatory RNA RydC by the 2 bacterial Hfq protein 3 4 5 Daniela Dimastrogiovanni1, Kathrin S. Fröhlich2,3, Katarzyna J. Bandyra1, Heather A. 6 Bruce1, Susann Hohensee1, Jörg Vogel2 and Ben F. Luisi1 7 8 9 1Department of Biochemistry, University of Cambridge, Tennis Court Road, 10 Cambridge CB2 1GA, U.K. 11 12 2Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider- 13 Str. 2, D-15, 97080 Würzburg, Germany 14 15 3 Current address: Department of Molecular Biology, Princeton University, Princeton, 16 NJ 08544, USA 17 18 *correspondence: [email protected] 19 20 21 22 Summary 23 24 Bacterial small RNAs (sRNAs) are key elements of regulatory networks that 25 modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is 26 an example of this class of riboregulators. Like many other sRNAs, RydC bears a 27 ‘seed’ region that recognises specific transcripts through base-pairing, and its 28 activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in 29 complex with E. coli Hfq at 3.48 Å resolution illuminates how the protein interacts 30 with and presents the sRNA for target recognition. Consolidating the protein-RNA 31 complex is a host of distributed interactions mediated by the natively unstructured 32 termini of Hfq. Based on the structure and other data, we propose a model for a 33 dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA 34 target. 35 36 37 Keywords: Hfq, small RNA, natively unstructured protein, protein-RNA recognition, 38 gene regulation 39 40 INTRODUCTION 41 42 The expression of genetic information is controlled and synchronised through 43 intricate regulatory networks. In bacteria, the control of gene expression post 44 transcription is mediated in part by small RNAs (sRNAs), and their contributions 45 enrich the computational complexity and repertoire of regulatory circuits (Beisel and 46 Storz, 2011). Bacterial sRNA are typically 50 to 300 nucleotides in length (Storz et 47 al., 2011), and each control expression of a distinct set of target mRNAs, which they 48 recognise with high specificity and apparent precision. One of the major facilitators of 49 sRNA activity in bacteria is the protein Hfq, which promotes pairing of an sRNA with 50 its target mRNA in solution (De Lay et al., 2013; Vogel and Luisi, 2011). Indeed, the 51 kinetics of target pairing seem an essential aspect of sRNA action in vivo, as many of 52 these sRNAs affect rates of translation or decay, either positively or negatively 53 depending on target and context (Desnoyers and Massé, 2012; Fröhlich and Vogel, 54 2009; Papenfort et al., 2013). It can be envisaged how Hfq acts as a catalyst for such 55 recognition in vivo, but the question naturally arises how sRNAs generally achieve 56 precision, accuracy and speed in producing their effects and avoid undesired off- 57 targets consequences. The key to understanding these fundamental processes of 58 sRNA-based regulation is to determine how RNAs are bound and presented by Hfq 59 and other effector proteins. 60 61 The previous crystal structures of truncated Hfq variants have provided clues as to 62 how the protein recognises short stretches of single stranded RNA. Hfq bears an + 63 fold that is the signature of the highly conserved family of Sm/Lsm proteins that 64 draws members from all domains of life (Kambach et al., 1999; Wilusz and Wilusz, 65 2013). Like other proteins of this extensive group, the bacterial Hfq self-assembles 66 into a ring-like architecture. Hfq of Escherichia coli and other eubacteria forms a 67 compact hexameric toroid that presents two structurally non-equivalent concave 68 surfaces for molecular recognition; these faces are referred to as the proximal and 69 distal faces, with the former exposing an N-terminal -helix (Link et al., 2009; 70 Schumacher et al., 2002). Crystallographic studies have identified interactions of 71 short RNA polymers with either of these two surfaces and have inferred sequence 72 preferences that have been corroborated and refined by mutagenesis and solution 73 binding studies (Link et al., 2009; Robinson et al., 2013; Sauer and Weichenrieder, 74 2011; Schumacher et al., 2002). According to these results, the proximal site interacts 75 preferentially with uridine-rich sequences, while the distal site favours the binding of 76 ARN or ARNN motifs (with R being a purine, and N any nucleotide) (Link et al, 77 2009; Sauer & Weichenrieder, 2011; Schumacher et al, 2002). In addition to the distal 78 and proximal faces, the torus-shaped Hfq hexamer bears a convex circumferential rim 79 that has recently been identified as a surface contributing to RNA binding (Sauer et 80 al., 2012; Zhang et al., 2013). Structural studies of the hetero-heptameric Sm 81 assembly from the mammalian splicing machinery have identified RNA binding 82 surfaces that share some similarities with the bacterial counterpart (Leung et al., 83 2011). 84 85 Bacterial sRNAs may recognize their cognate target mRNAs in different ways. In 86 some sRNAs, a target-recognition region is presented at or near the 5’ end (Papenfort 87 et al., 2010). For another subset of sRNAs, several pairing regions are non-continuous 88 and each can independently bind distinct targets (Beisel and Storz, 2011; Shao et al., 89 2013). In both cases, the sRNA recognition site (referred to as the ‘seed’) and the 90 target can have imperfect complementarity of various lengths with interactions being 91 as short as 6 base-pairs, as seen for example in the SgrS/ptsG mRNA pair from E. coli 92 (Kawamoto et al., 2006). To fully understand the mechanism of sRNA mediated 93 regulation, it is important to address the questions of how the intricate RNA folds are 94 recognised, how the seeds are presented, and how the pairing of sRNAs with mRNAs 95 is facilitated. As the sRNAs, mRNAs and their complexes can all be remodelled upon 96 binding Hfq, the rules for recognition are likely to be highly dependent on context. 97 98 Here, we describe the structure of the sRNA RydC in complex with the full-length 99 Hfq protein of E. coli. Previous studies demonstrated that RydC is involved in biofilm 100 regulation (Bordeau and Felden, 2014) as well as the control of membrane stability 101 through the positive regulation of an isoform of the cfa mRNA encoding 102 cyclopropane fatty acid synthase (Fröhlich et al., 2013). RydC is proposed to have a 103 pseudoknot fold that exposes a 5’ seed sequence, and the in vivo stability of RydC 104 requires Hfq (Antal et al., 2005; Bordeau and Felden, 2014; Fröhlich et al., 2013). 105 The crystal structure of the RydC-Hfq complex, together with biochemical and in vivo 106 data, define key interactions and suggests a hypothetical model for how the sRNA and 107 mRNA targets might be matched through Hfq binding. 108 109 RESULTS 110 111 RydC interaction with Hfq and pseudoknot structure are essential for its stability in 112 vivo 113 114 Despite its low abundance in comparison with other sRNAs under standard growth 115 conditions, RydC has been repeatedly recovered with the Hfq protein in pulldown 116 assays (Chao et al., 2012; Sittka et al., 2008; Zhang et al., 2003). A distinctive feature 117 of RydC is its predicted pseudoknot fold which is highly conserved, and whose 118 disruption by point mutations renders the RNA unstable in vivo (Fröhlich et al., 119 2013). A double mutant of RydC (G37C and G39C within helix 1; hereafter, RydC- 120 S1) was predicted computationally to form a distinct structure with one short stem- 121 loop at the 5’ end and a strong terminator hairpin at the 3’ end (Fig. 1a). 122 123 To assess the molecular mechanism underlying the intrinsic instability of the RydC- 124 S1 mutant we compared its turnover rate to the wild-type RNA in the presence and 125 absence of the chaperone Hfq (Fig. 1b/c). To this end, both RydC and RydC-S1 were 126 expressed in Salmonella under the control of the constitutive PL promoter from high- 127 copy plasmids. At exponential growth (OD600 of 1), transcription was stopped by the 128 addition of rifampicin, and RNA levels were monitored at various time-points after 129 treatment. As previously shown (Fröhlich et al., 2013), RydC is very stable in wild- 130 type cells (half-life (t1/2) >32 min), while RydC-S1 decayed rapidly (t1/2 ~2 min). In 131 contrast, the half-life of RydC is already markedly reduced in hfq mutant cells (t1/2 ~4 132 min) and there is some further acceleration of the decay rate for RydC-S1, matching 133 that of RydC in the absence of Hfq. This observation led us to assume that RydC 134 stability depends on both the integrity of the pseudoknot fold and the ability to 135 associate with the Hfq protein. Both the wild type RydC and RydC-S1 mutant bind 136 Hfq in electrophoretic mobility shift assays with in vitro synthesized RNA although 137 the affinity of the mutant is lower than for the wild type RydC (Figure 1 – figure 138 supplement 1a). Competition experiments suggest that the binding sites for the RydC 139 and RydC-S1 overlap at least partially (Figure 1 - figure supplement 1b). This 140 suggests that the destabilisation in vivo might result mostly from the integrity of the 141 pseudoknot fold, but to some degree the Hfq binding alone may inhibit the 142 degradation of RydC from initiating in single-stranded regions as suggested for other 143 sRNAs (Moll et al., 2003; Saramago and Arraiano et al.
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