An RNA-dependent mechanism for transient expression of bacterial translocation filaments

Abstract

The prokaryotic RNA chaperone Hfq mediates sRNA–mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5′ UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.

A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis

“…Lately, it was reported that in the E. coli enterocyte effacement locus (LEE) Hfq plays a repressive and CsrA an activating role on sepL mRNA [52]. However, since the motif required for CsrA binding overlaps the second Hfq interaction site, binding of both chaperones is mutually exclusive.…”

After the Fact(or): Posttranscriptional Gene Regulation in Enterohemorrhagic Escherichia coli O157:H7

“…6E). sepL is the first gene in LEE4, and several studies indicated that expression of the entire operon is dependent on its transcription and translation (111,(124)(125)(126) (Fig. 6E).…”

“…The expression and activity of Ler, GrlA, and GrlR are a hub of regulation at all levels within the cell. A recent study provided evidence that Hfq binds multiple RNA transcripts throughout the LEE, including LEE1 and grlRA (111). Although the authors extensively characterized the involvement of Hfq in expression of LEE4 (detailed below), no data describing the specific mechanism of Hfq-dependent regulation of Ler or GrlA expression were reported in that study.…”

“…The nascent sepL 5= UTR forms a structure that sequesters the SD sequence and thereby prevents translation (111). CsrA binds the sepL leader sequence to expose the SD sequence and promote translation of the entire operon (111,113). Hfq and the sRNA Spot42 also bind sepL mRNA and repress translation (111).…”

Here, there, and everywhere: How pathogenicEscherichia colisense and respond to gastrointestinal biogeography

“…The activity of these sRNAs on grlRA is dependent on Hfq, a bacterial RNA chaperone that mediates interactions between sRNA and mRNA. Hfq was recently found to bind to LEE4 and LEE5 operons, suggesting a way for sRNAs to influence T3SS regulation (Wang et al, ). There is also evidence of regulation by sRNAs in other T3SS‐expressing pathogens, such as Yersinia pseudotuberculosis , Yersinia pestis , and S .…”