Positive regulation by small RNAs and the role of Hfq

Abstract

Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5' leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)(4) Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 degrees C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA*mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA*mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.

Fig. 1.

sRNA activation of rpoS translation requires Hfq. The rpoS mRNA leader forms an inhibitory secondary structure that is relieved by Hfq-dependent DsrA, RprA, or ArcZ binding.

Fig. 2.

rpoS::lacZ fusions activated by DsrA, RprA, and ArcZ. (A) A summary of in vitro results from (23) showing the importance of rpoS leader length and an (AAN)₄ element (red box) for the action of Hfq. The numbers indicate the nucleotide at the 5′ end of the rpoS leader RNA, relative to the natural start; the in vitro RNAs used both previously and in this work extended 12 nt into the ORF. The “double-mutant” construct had the properties of the (AAN)₄ mutant. Structure of the 5′ leader and sequence of the mutations in the A-rich elements are shown in Fig. S2. (B) The rpoS leader constructs carrying the truncations and mutations described in Fig. 2A were fused to lacZ to create translational fusions under the control of the arabinose-inducible P_BAD promoter; the (AAN)₄ and A₆ point mutations were introduced into the full-length fusion rather than the long fusion shown in Fig. 2A. The specific strains are described in Table S1. (C) sRNA activation of rpoS leader fusions. Strains containing the vector pBRplac (black bars) or plasmids overexpressing DsrA, RprA, or ArcZ, were grown in LB containing arabinose and IPTG at 37 °C to stationary phase before ß-galactosidase activity was measured. (D) sRNA activation of rpoS leader fusions in an hfq^- background. Same as in C, with hfq::cat derivatives; white bars contain vector plasmid. Note that y axis values are significantly less in D than in C.

Fig. 3.

Hfq binds specifically to full-length, but not processed, ArcZ. (A) ArcZ121 RNA was titrated with Hfq and subjected to native gel electrophoresis. Shifted bands are interpreted as ArcZ121 bound by one (A•H), two (A•H2), or three (A•H3) Hfq hexamers. These transitions were fit as shown in Fig. S6 to give K_H1 = 0.09 μM Hfq₆ and K_H2 = 0.45 μM Hfq₆. (B) ArcZ56 was analyzed as for A; only a small proportion of the counts migrated in the observed bands. The rest formed a smear of high molecular weight complexes.

Fig. 4.

sRNAs binding the rpoS leader at 37 °C. (A) sRNA titrations of the long rpoS leader in the absence of Hfq. DsrA (blue circles), RprA (orange squares), ArcZ56 (brown diamonds), and ArcZ121 (red triangles) were mixed with the long rpoS leader RNA and subjected to native gel electrophoresis and the formation of a complex calculated as described in Materials and Methods. (B) As for A, with the addition of Hfq.

Fig. 5.

Summary model. The population of open (activated) rpoS leader in the presence of sRNAs was simulated from the in vitro binding data [Table 1, (23)]: DsrA at 37 °C (dark blue), DsrA at 25 °C (light blue), and ArcZ (brown). Solid curves, no Hfq; dashed curves, with Hfq. Vertical dashed lines show the expected fraction of translatable rpoS leader when sRNAs are present at either endogenous or overexpressed levels. (Top) sRNA activation of the WT rpoS leader; (Bottom) sRNA activation of the (AAN)₄ mutant rpoS leader. It was assumed that the mutation does not affect sRNA binding, and that the presence of Hfq improves sRNA binding to the mutant by ∼1.5-fold, similar to the kinetic behavior reported in ref. .