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Contents
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An RNA Regulator for Avoiding Sugar-phosphate Stress
A major class of bacterial non-coding RNAs conducts posttranscriptional regulatory functions by basepairing with target mRNAs and altering their translation or stability. Thus far, all of the members of this class in E. coli, such as the RNA described here, SgrS, bind to and require an RNA chaperone protein called Hfq to perform their regulatory functions. SgrS is encoded as a 227-nucleotide (nt) transcript in a region between two protein coding genes. The first clue to its physiological role came with the observation that cells overexpressing SgrS could not grow using glucose as the only energy source, but were capable of using many other sugars. A previous study from the laboratory of Hiroji Aiba (Nagoya University, Japan) (Morita T. et al. J Biol Chem 278: 15608–14, 2003) had documented an unexplained posttranscriptional regulatory phenomenon involving the ptsG mRNA, which encodes the major glucose transporter in E. coli. The PtsG protein transports glucose through the cytoplasmic membrane of the bacterial cell, phosphorylating it in the process (Figure 1, part A). Aiba and colleagues found that in cells that accumulated excess glucose-phosphate or the glucose-phosphate analog α-methyl glucoside (αMG)-phosphate, the ptsG mRNA became very unstable. We hypothesized that if SgrS were causing the ptsG transcript to become unstable, cells overexpressing SgrS might not be able to transport enough glucose to use it as a carbon source for growth. Non-metabolizable sugar-phosphate molecules are known to be growth inhibitory or sometimes lethal by mechanisms that are not completely understood. Normal E. coli cells that are exposed to αMG transport and phosphorylate this molecule via PtsG, but cannot metabolize the phosphorylated sugar further. As a result, their growth is transiently inhibited. On the other hand, growth in sgrS-negative mutant cells is more severely inhibited upon exposure to αMG, suggesting that (1) a greater amount of toxic phosphorylated sugar is produced and (2) SgrS is important to reduce the level of phosphorylated sugar, which helps reduce sugar-phosphate stress. The levels of SgrS RNA and ptsG mRNA were examined and were found to be reciprocal. In the absence of αMG, we could not detect SgrS, but the ptsG mRNA was present. Within 5 minutes after cells were exposed to αMG, large amounts of SgrS RNA appeared, and the ptsG message had disappeared. However, in sgrS mutant cells, the ptsG mRNA did not disappear. Since other Hfq-binding non-coding RNAs in E. coli act by basepairing with their targets, we examined the SgrS and ptsG RNAs for regions of complementarity. SgrS has the potential to basepair with the ptsG mRNA in the 5´ untranslated region in an area that overlaps with the ribosome binding site. This suggests that SgrS:ptsG mRNA basepairing may inhibit translation of the ptsG mRNA and lead to its rapid degradation by inhibiting ribosome binding, a mechanism that has been described for other non-coding RNAs in E. coli. Figure 1. A model for the cellular response to glucose-phosphate stress where SgrR and SgrS function to modulate the transporter for glucose. A) The PtsG transporter brings glucose into the cell, phosphorylating it in the process. B) Accumulated glucose-phosphate is proposed to interact with the regulatory protein SgrR to stimulate transcription of the regulatory RNA SgrS, which binds to the RNA chaperone Hfq. C) SgrS pairs with the mRNA encoding PtsG, leading to degradation of the small RNA and the ptsG mRNA. This downregulates further transport of glucose into the cell, limiting the accumulation of toxic sugar-phosphates. The pattern of αMG-inducible SgrS synthesis indicated that expression of SgrS was controlled by one or more factors that could respond to increased levels of sugar-phosphates. In bacteria, genes related in function are often found in close proximity to one another; frequently, a gene encoding a regulatory protein is positioned near the genes it regulates. The sgrR gene, divergently transcribed from sgrS, is present only in bacterial genomes that contain SgrS. Furthermore, the SgrR protein was predicted to contain an N-terminal DNA binding domain similar to those of other transcription factors. SgrR is in fact required for SgrS synthesis. In addition to its DNA-binding domain, SgrR also contains a domain similar to sugar-binding domains of other proteins. We propose that SgrR itself is the sensor of intracellular sugar-phosphates and the activator of SgrS synthesis.
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growing awareness of the number and importance of non-coding RNA molecules in
the regulation of gene expression in both bacteria and eukaryotes has driven
research into RNA-mediated regulatory phenomena in recent years. In bacteria,
most notably Escherichia coli, several global searches have revealed
that the number of genome coding sequences for non-coding RNAs is between 1%
and 2% of the number of coding sequences for proteins. Of the non-coding RNAs
that have been characterized in E. coli, many are involved in regulating
gene expression under specific stress conditions, such as low temperature, iron
starvation, and acid stress. In this study, we characterized a novel non-coding
RNA in E. coli that responds to disruptions in sugar metabolism that
alter flux through the glycolytic pathway. This non-coding RNA, SgrS, is necessary
to prevent the accumulation of non-metabolizable phosphosugars, which are toxic
to cells.