Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

Abstract

The Escherichia coli gal operon has the structure Pgal-galE-galTgalK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the downregulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rhomediated transcription termination at the end of galT.D uring the exponential growth phase of Escherichia coli cells, transcription termination at the end of each cistron of the gal operon operates with less than 100% efficiency; a certain proportion of the transcription initiated from the two promoters of the gal operon terminates at the end of each constituent gene, galE, galT, galK, and galM, of the operon (1). The termination efficiencies measured at the end of each cistron are 16%, 29%, 65%, and 71%, respectively (1). Transcription termination at the end of each cistron generates four mRNA species: mE1, mT1, mK1, and mM1 (Fig.

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria

“…In E. coli, the 109 nt long Spot 42 sRNA encoded by the spf gene is known to downregulate expression of galK, the third gene of the galactose operon galETKM, thereby inhibiting GalK production (Møller et al, 2002). Recent studies suggested that Spot 42 regulates galK expression at two levels: RNA degradation and enhancement of Rho-dependent transcription termination at the galT-galK junction (Wang et al, 2015b). To explain how Rho assures termination at the end of the galT gene, Wang et al (2015b) proposed that the binding site of Spot 42 on the galT-galK junction RNA overlaps with a putative rut site.…”

“…Recent studies suggested that Spot 42 regulates galK expression at two levels: RNA degradation and enhancement of Rho-dependent transcription termination at the galT-galK junction (Wang et al, 2015b). To explain how Rho assures termination at the end of the galT gene, Wang et al (2015b) proposed that the binding site of Spot 42 on the galT-galK junction RNA overlaps with a putative rut site. Spot 42 binding enhances the disassembly of the ribosome at the stop codon of galT and exposes the rut site.…”

“…Spot 42 binding enhances the disassembly of the ribosome at the stop codon of galT and exposes the rut site. Rho would finally catch up with the RNAP transcribing downstream DNA and terminate transcription (Wang et al, 2015b).…”

Carbohydrate Utilization in Bacteria: Making the Most Out of Sugars with the Help of Small Regulatory RNAs

“…However, Spot 42 also alters target mRNA levels (75). In-depth study of the galETKM operon revealed that Spot 42 stimulates Rho-dependent transcription termination at the galT-galK junction re-capitulating observations for ChiX (56,81). A non-canonical mechanism of Spot 42 action was observed for the sdhCDAB mRNA encoding succinate dehydrogenase (82).…”

The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

“…Recently, Wang et al showed that Spot 42 represses expression of galK through direct binding to the 5 end of the galK mRNA , and also mediates transcription termination of galT in the galT-galK junction. 13 Beisel and Storz demonstrated with microarray analysis and reporter fusions that Spot 42 plays a broader role in metabolism by regulating at least 14 operons. 14 These operons contain a number of genes involved in uptake and catabolism of non-favored carbon sources.…”