An analysis of cis-encoded and trans-encoded sRNA metabolism in Escherichia coli K-12
Marshburn, Sarah Evelyn
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In order to survive, bacteria must be able to adapt and persist in a variety of poor growth conditions. To do this, cells undergo physiological and morphological changes that are influenced by changes in gene expression. Although proteins dominate the regulatory landscape, the identification and functional characterization of small RNAs (sRNAs) in diverse bacterial lineages has shown that sRNAs are widely used as potent effectors of post-transcriptional regulation that mediate and integrate numerous physiological responses. Typically, sRNAs affect, either positively or negatively, the translation and/or stability of their target mRNA(s). It is generally believed that most sRNAs function stoichiometrically, i.e. one sRNA molecule regulates one target mRNA molecule because sRNA:target degradation is coupled. Therefore, sRNA decay is, in some respects, a consequence of function. Although this intimate connection between metabolism and function exists, sRNA metabolism has not been comprehensively or deeply investigated. To further understand sRNA metabolism in vivo, this study examined the metabolism of two trans-encoded sRNAs, ArcZ and RprA, and two cis-encoded sRNAs, GadY and SibC. Generally, sRNAs are classified based on the genomic position of the sRNA gene relative to the target gene as this has structural and mechanistic implications. Cis-encoded sRNA genes overlap, are antisense to their target gene(s) and have the potential to form long, perfect duplex structures with their target(s). In contrast, trans-encoded sRNAs genes do not overlap their target gene(s) and typically form short, imperfect duplexes with their targets. To assess global ribonuclease involvement in sRNA metabolism, we examined an isogenic set of mutants containing loss-of-function alleles of all the known major cytoplasmic endoribonucleases and exoribonucleases of E. coli K-12. The results of this study revealed a global involvement of both RNase E and polynucleotide phosphorylase (PNPase) in the metabolism of all four sRNAs. Surprisingly, RNase P appeared to participate in the metabolism of RprA, GadY and SibC, whereas RNase G, RNase III and RNase II were necessary for the metabolism of GadY and SibC. When taken together, our results suggested the existence of multiple, complex metabolic pathways that may involve hierarchical cleavage events, as well as, target-dependent and target-independent metabolic pathways.
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