GlmY and GlmZ: a hierarchically acting cascade composed of two small RNAs
Göpel, Yvonne
Dissertation
Angenommen am:
2013-10-02
Erschienen:
2014-09-10
Betreuer:
Görke, Boris PD Dr.
Gutachter:
Ficner, Ralf Prof. Dr.
Gutachter:
Walter, Lutz Prof. Dr.
Gutachter:
Pöggeler, Stefanie Prof. Dr.
Gutachter:
Hoppert, Michael PD Dr.
Gutachter:
Daniel, Rolf PD Dr.
Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0022-5F6E-2
Dateien
Name:
Dissertation Yvonne Göpel.pdf
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11,9 MB
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PDF
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Zusammenfassung
Englisch
In Escherichia coli and other enterobacteria, the homologous small RNAs (sRNAs) GlmY and GlmZ act in a hierarchical manner to feedback control expression of key enzyme glucosamine-6-phosphate (GlcN6P) synthase GlmS. Enzyme GlmS catalyzes formation of GlcN6P, which is the rate limiting reaction in the pathway of cell wall biosynthesis. Only sRNA GlmZ can activate the glmS mRNA by base-pairing, which releases the ribosomal binding site and allows synthesis of GlmS. The second sRNA GlmY acts indirectly to activate glmS by stabilizing GlmZ in a process that involves protein YhbJ. However, the molecular mechanism remained elusive. As sRNAs GlmY and GlmZ are crucial in maintaining essential cellular functions and are thus tightly controlled, we aimed to gain insight into the complex regulation of these sRNAs at the level of biosynthesis and decay. First, we investigated control of glmY and glmZ transcription in several species using Yersinia pseudotuberculosis, Salmonella thyphimurium and E. coli as representatives. Three different promoter architectures were observed: (I) In Y. pseudotuberculosis expression of both sRNAs is driven solely from σ54-promoters; (II) perfectly overlapping σ70- and σ54-dependent promoters control expression of glmY in E. coli and of both sRNA genes in S. thyphimurium; (III) in contrast, glmZ of E. coli is constitutively expressed from a σ70-promoter. These results suggest that the glmY/Z system is in evolutionary transition from σ54- to σ70-dependency in a subset of species. Moreover, the σ54-dependent promoters are activated by the two component system GlrK/GlrR and rely on integration host factor IHF for activity. Further, we found that acetylation of YhbJ is required for full activity of the σ54-promoter of glmY in E. coli and that sirtuin deacetylase CobB drastically reduces promoter activity. In sum, GlmY and GlmZ seem to compose a regulon dependent on σ54, GlrK/GlrR and IHF in the majority of Enterobacteriaceae. Second, we clarified the molecular mechanism of signal transduction within the GlmYZ cascade. We demonstrated that YhbJ is a novel RNA-binding protein that binds GlmY and GlmZ with high affinity and switches its sRNA binding partners depending on the intracellular GlcN6P level. Under conditions of ample GlcN6P, YhbJ preferably binds GlmZ and recruits its processing machinery by protein-protein interaction with the major endoribonuclease RNase E. GlmZ is inactivated by RNase E and subsequently degraded. Upon GlcN6P depletion, GlmY accumulates and sequesters YhbJ thereby counteracting processing of GlmZ. In analogy to regulated proteolysis we renamed protein YhbJ to RapZ as acronym for RNase adaptor protein for sRNA GlmZ; thus, GlmY acts as an anti-adaptor decoy for protein RapZ. Even though GlmY and GlmZ are highly similar in sequence and structure, both sRNAs act by distinct mechanisms. While GlmZ is a base-pairing sRNA that depends on RNA chaperon Hfq for functionality and stability, GlmY acts solely by protein-binding and does not require Hfq. Moreover, GlmZ is processed by RNase E in a RapZ-dependent manner, whereas GlmY is not. Hence, we exploited GlmY and GlmZ as model system to study the molecular requirements for Hfq-binding and processing by RNase E. We found that the entire 3’ end of GlmZ is required for high affinity binding by Hfq and Hfq-dependent stabilization in vivo. In contrast, the lateral bulge within the central stem loop of GlmZ is of prime importance for recognition by RNase E. In sum, our findings reveal an unprecedented mechanism controlling the activity of a small RNA at the level of its turnover. This mechanism involves the novel RNase adaptor protein RapZ, which might be the first of similar proteins conferring substrate specificity to a general ribonuclease.
Keywords: GlmY, GlmZ, sRNA, RapZ, glucosamine-6-p homeostasis, RNA degradation, RNase E, RNase adapter protein
