Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs and mRNAs in live bacterial cells

Abstract

Hfq binds broadly to cellular mRNAs and sRNAs [13][14][15] , in line with its main biological 51 functions. Hfq can bind RNAs through multiple interfaces of its homohexameric structure. The 52 surface containing the N-terminal α-helices is referred as the "proximal face" of the Hfq hexamer, 53whereas the opposite surface is referred as the "distal face", and the outer ring as the "rim" (Figure 54 1a). The proximal face binds preferably U-rich sequences, and the distal face prefers A-rich 55 sequences, with the exact motif of the A-rich sequence depending on the species [16][17][18][19][20] . The rim 56 can also interact with UA-rich RNAs through the patch of positively charged residues 21-23 . Finally, 57 the unstructured C-terminal end of Hfq can also interact with certain RNAs to promote the 58 exchange of RNAs 18,21,24 . The most refined model describing the interactions between Hfq and 59 sRNAs/mRNAs has sorted sRNAs into two classes 25 . The proximal face of Hfq is generally 60 important for binding of the sRNAs through their poly-U tail of the Rho-independent terminator. 61Class I sRNAs (such as RyhB and DsrA) use the rim as the second binding site, whereas class II 62 sRNAs (such as ChiX and MgrR) use the distal face as the second binding site 25 . In addition, the 63 preferred target mRNAs of the two classes of the sRNA are proposed to have the complementary 64 binding sites on Hfq, i.e. class I sRNA-targeted mRNAs binding to the distal face, and class II 65 sRNA-targeted mRNA binding to the rim, in order to efficiently form sRNA-mRNA complexes 25 . 66As for many other RBPs, the functions of Hfq are facilitated by its interactions with other essential 67 protein factors. Particularly, RNase E, the key ribonuclease for processing and turnover of 68 ribosomal RNA (rRNAs) and mRNAs, is known to interact with Hfq through its C-terminal scaffold 69 region [26][27][28] . The Hfq-RNase E interactions can promote degradation of the sRNA-targeted 70 mRNA 28-31 . 71 While Hfq is an abundant RBP in bacterial cells 32,33 , it is still considered to be limiting, given 72 the abundance of cellular mRNAs and sRNAs. Particularly, in vitro studies on specific sRNAs 73 demonstrate that Hfq binds RNAs tightly with a dissociation constant of nM, and the Hfq-RNA 74

The RNA-binding protein Hfq assembles into foci-like structures in nitrogen starved Escherichia coli

“…As shown in Fig. 3A and 3B, in bacteria at N+ and N-, the D* values of Hfq molecules were largely similar (though we note that our D* values were ~6-fold lower than those measured by Persson et al (28) with Dendra2tagged Hfq in exponentially growing E. coli in lysogeny broth, our measurements are largely consistent with those measured by Park et al (29) with mMaple3 tagged Hfq in E. coli growing in defined in minimal media). Of note, the graph in Fig.…”

“…( 28 ) with Dendra2-tagged Hfq in exponentially growing E. coli in lysogeny broth, our measurements are largely consistent with those measured by Park et al . ( 29 ) with mMaple3-tagged Hfq in E. coli growing in defined in minimal medium). Of note, the graph in Fig.…”

Kinetic modeling reveals additional regulation at co-transcriptional level by post-transcriptional sRNA regulators

“…the plasmid expression of the target mRNAs did not cause Hfq to become a limiting factor. In addition, a recent study reveals that sRNAs can effectively gain access to Hfq even if it is already occupied by mRNAs in vivo (Park et al, 2021), further arguing against Hfq becoming the limiting factor in our measurements.…”

“…Here, using single-molecule localization microscopy (SMLM), we confirmed the unbiased localization for endogenously expressed SgrS and RyhB (Figures 7B-7E). In addition, the chaperone protein, Hfq, was observed to diffuse freely into the nucleoid region (Park et al, 2021;Persson et al, 2013) and to bind to nascent transcripts (Kambara et al, 2018). It is likely that at least part of the Hfq binding to the nascent transcripts is mediated by sRNAs.…”

Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

“…Here, using single-molecule localization microscopy (SMLM), we confirmed the unbiased localization for these two sRNAs under our experimental conditions (Figure 7). In addition, the chaperone protein, Hfq, was observed to diffuse freely into the nucleoid region using single-particle tracking ( 71 , 72 ) and to bind to the nascent transcripts in a recent study using Chip-seq ( 73 ). It is likely that at least part of the Hfq binding to the nascent transcripts is mediated by sRNAs.…”