Signal perception and transduction by small RNAs GlmY and GlmZ

Small regulatory RNAs (sRNAs) are a well-established class of gene regulators controlling numerous physiological circuits in bacterial cells. However, how sRNAs themselves are regulated is less understood. The GlmY/GlmZ sRNA cascade of Escherichia coli represents an example of a complex sRNA-regulated circuit with many input functions. GlmY and GlmZ are homologous sRNAs that jointly attune synthesis of enzyme glucosamine-6-phosphate (GlcN6P) synthase GlmS to the concentration of its product GlcN6P. Of both sRNAs exclusively sRNA GlmZ can base-pair with an inhibitory stem loop structure within the glmS mRNA that masks the ribosome binding site. Base-pairing activates synthesis of GlmS which catalyzes formation of GlcN6P, an essential precursor for cell wall biosynthesis. However, GlmZ is subject to processing by RNase E which removes the base-pairing nucleotides and initiates degradation of GlmZ. Our recent data show that GlmZ per se is not a substrate for RNase E and that processing of GlmZ in vivo and in vitro requires protein RapZ. RapZ turned out to be a novel type of sRNA binding protein that binds GlmZ and targets it to processing by a mechanism that involves physical interaction with RNase E. The second sRNA GlmY, which accumulates upon intracellular GlcN6P depletion, acts indirectly to activate glmS expression. GlmY is a decoy RNA and sequesters RapZ, thereby preventing processing of GlmZ. Thus, RapZ is an adaptor and GlmY an anti-adaptor in the regulated turnover of the base-pairing sRNA GlmZ. This novel mechanism explains how specificity in the controlled decay of a particular sRNA can be achieved. Important questions concerning the GlmY/GlmZ/RapZ regulatory circuit remain open and shall be investigated in the proposed project. Firstly, it shall be addressed how RapZ mechanistically acts in the controlled turn-over of GlmZ. It is conceivable that RapZ is an allosteric activator of RNase E. Alternatively it might act by inducing structural changes in GlmZ, thereby converting it to a substrate that can be recognized by RNase E. Secondly, it shall be clarified how the activity of the GlmY/GlmZ circuit is controlled by GlcN6P. Our preliminary data suggest that GlmY activity is controlled by direct binding of GlcN6P. This opens the intriguing possibility that GlmY represents the first example for a metabolite-binding autonomously encoded sRNA. Taken together, the proposed project is anticipated to yield insight into the mechanistic basis of how a globally acting RNase can be directed towards a specific RNA substrate and how a specific signal is sensed by a sRNA at the post-transcriptional level.

E. coli and related Gram-negative bacteria possess a cell envelope consisting of peptidoglycan and two membranes, which provides protection against environmental stresses. Synthesis of glucosamine-6-phosphate (GlcN6P) by enzyme GlmS represents the first and rate-limiting step in the cell envelope synthesis pathway. In order to ensure continuous cell envelope synthesis, GlcN6P homeostasis is required, which is achieved through a post-transcriptional regulatory circuit composed of small RNAs (sRNAs) GlmY and GlmZ and the RNA-binding protein RapZ. GlmZ activates glmS translation by base-pairing. When GlcN6P is ample, GlmZ is bound by adaptor protein RapZ and inactivated by cleavage through recruitment of RNase E. Decreasing GlcN6P concentrations provoke accumulation of the homologous sRNA GlmY, which acts as decoy and sequesters RapZ thereby counteracting GlmZ decay. First, we dissected the molecular features underlying the different functions of the two homologous sRNAs. This analysis led to the discovery of an RNA aptamer that can be used as tool to produce RNAs of interest in their 5-monophosphorylated forms on demand in vivo, which might have interesting applications. The crystal structure of RapZ was solved revealing a novel type of RNA-binding protein, which putatively evolved from re-purpose of a glycolytic enzyme. The active form of RapZ corresponds to a tetramer, which allosterically activates the likewise tetrameric RNase E in an encounter complex to cleave sRNA GlmZ that is presumably sandwiched between the two tetramers. In addition, RapZ also represents the sought GlcN6P sensor: RapZ binds GlcN6P with high affinity and rapZ mutants are blind for this metabolite. Upon GlcN6P depletion, RapZ stimulates a transcription factor through interaction to increase GlmY expression levels. Thus, RapZ controls production of its own decoy GlmY, which subsequently sequesters the available RapZ protein into long-lived ribonucleoprotein complexes, thereby counteracting GlmZ decay. Finally, we assessed whether this regulatory system could be exploited for antimicrobial chemotherapy. Antibiotics are known, which act via inhibition of the GlmS enzyme, but are not used against Gram- negative bacteria due to their limited potency. We find that this intrinsic resistance is mediated by the GlmY/RapZ/GlmZ system, which overcomes inhibition of GlmS by strong up-regulation of glmS expression. Thus, compounds inhibiting this regulatory cascade, such as non-metabolizable GlcN6P analogs, represent attractive adjuvants that can significantly amplify the potency of antibiotics targeting enzyme GlmS.