Regulation of glucosamine-6-phosphate synthase synthesis by a hierarchical acting cascade composed of two small regulatory RNAs in Escherichia coli . (2010)

Amino sugars are essential for the biosynthesis of the bacterial cell wall and of lipopolysaccharides. The enzyme glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine-6-phosohate (GlcN6P) from fructose-6-phosphate and glutamine, which is the first committed step in the biosynthesis of amino sugars. In Escherichia coli, GlmS is encoded together with enzyme GlmU in the bi-cistronic glmUS operon. While the enzymatic activity of GlmU is needed all the time, GlmS is only required, when no external amino sugars are available. Due to the fact that both genes are cotranscribed, it always was a mystery how differential expression of glmU and glmS could be achieved. This work solves this mystery. Following transcription, the glmUS co-transcript is cleaved within the stop-codon of glmU by RNase E, yielding a monocistronic glmS transcript. The amount of the glmS-transcript is regulated by a feedback mechanism in response to an intracellular limitation of GlcN6P. This regulation relies on the two small RNAs GlmY and GlmZ. GlmY and GlmZ are homologous sRNAs, both in sequence and in secondary structure, and act in a cascade to activate glmS expression. When the GlcN6P level decreases, GlmY accumulates and subsequently counteracts processing of GlmZ by RNase E. Only the unprocessed form of GlmZ is able to basepair with the glmS transcript. GlmZ basepairs with the left half-site of a stem-loop structure assisted by Hfq. This prevents access of ribosomes to the Shine-Dalgarno sequence. This interaction destroys the inhibitory stem-loop, thereby allowing efficient translation of glmS. In addition, the data indicate that the protein YhbJ might might play a role in this process. In a ΔyhbJ mutant, processing of GlmZ is abrogated and glmS expression is strongly induced. Therefore, it appears feasible that YhbJ is an intermediary factor in the GlmY/GlmZ cascade, which acts between GlmY and GlmZ and regulates GlmZ p rocessing in a GlmY-dependent manner. Both expression and abundance of GlmY, which acts at the top of this regulatory cascade, is extensively regulated. GlmY is continuously targeted for polynucleotide phosphorylase dependent degradation by poly(A)polymarase dependent polyadenylation. This removes active GlmY species from the cell and keeps the cascade sensitive to the GlcN6P signal. Gene glmY is expressed from two overlapping σ70- and σ54-dependent promoters. Both promoters initiate transcription at the same nucleotide. Therefore, identical GlmY species are generated from both promoters. The σ70-dependent promoter is active mainly during the exponential growth phase, while activity of the σ54-dependent promoter increased during transition to stationary phase. The σ70-dependent promoter appears to be constitutively active. In contrast, the σ54-dependent promoter subject to regulation by the GlrR/GlrK two component system and YhbJ. GlrR is a σ54-dependent activator protein and is shown to bind to three specific binding sites of the consensus sequence TGTCN10GACA, which are present upstream of the glmY promoter region. The activity of the σ54-dependent promoter depends on the presence of GlrR. The activity of this promoter is also reduced in a ΔyhbJ mutant. This effect is independent from GlmY and GlmZ, but the mechanism of YhbJ-dependent modulation of glmY σ54-promoter activity is unknown. Activity of the glmY-promoters is not altered in response to GlcN6P limitation. Therefore, the GlcN6P signal must be sensed post transcription of glmY. In summary, this work shows how differential expression of the bi-cistronic glmUS operon is achieved by post-transcriptional regulation of glmS expression by two homologous sRNAs, GlmY and GlmZ. These sRNAs act in a cascade and are the first example for hierarchicall y acting sRNAs. In addition, this work presents the first example of overlapping σ70- and σ54-dependent promoters that initiate transcription at the same nucleotide.