Abstract
OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear. We have investigated RprA and OxyS interactions with Hfq using biochemical and biophysical approaches. In particular, we have obtained the molecular envelopes of the Hfq-sRNA complexes using small-angle scattering methods, which reveal key molecular details. These data indicate that Hfq does not substantially change shape upon complex formation, whereas the sRNAs do. We link the impact of Hfq binding, and the sRNA structural changes induced, to transcript stability with respect to RNase E degradation. In light of these findings, we discuss the role of Hfq in the opposing regulatory functions played by RprA and OxyS in rpoS regulation.
Highlights
RpoS is a σ factor of stationary growth phase in Escherichia coli that functions as a master regulator, activating a plethora of genes involved in general stress response
Hfq interacts promiscuously with many RNAs containing an AU-rich sequence adjacent to a stem–loop (Brescia et al 2003; Moll et al 2003). This lack of binding specificity can result in multiple Hfq hexamers binding to a single RNA, as is the case for the small noncoding RNAs (sRNAs) RprA and OxyS
For OxyS, the data gave a Kd of 5.6 nM for complex I and a Kd of 53 nM for complex II. While these values are similar to the dissociation constants previously determined for complex I of both RprA:Hfq and OxyS:Hfq and complex II of RprA:Hfq
Summary
RpoS is a σ factor (σs) of stationary growth phase in Escherichia coli that functions as a master regulator, activating a plethora of genes involved in general stress response (for review, see Repoila et al 2003). This is caused either by Hfq changing the structure of the RNA, leading to the exposure of novel RNase cleavage sites (Zhang et al 2002), or by targeting specific mRNAs for degradation through the formation of specialized ribonucleoprotein complexes, comprising Hfq, RNase E, and an sRNA (Morita et al 2005; Bandyra et al 2012) This complex is believed to assemble on the C-terminal region (CTR) of RNase E (amino acids 530–1061) and acts to guide RNase E on to the target mRNA for endonucleolytic degradation. We assess how the interaction with Hfq affects the susceptibility of OxyS and RprA to RNase E attack
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