6S RNA Regulation of pspF Transcription Leads to Altered Cell Survival at High pH

Abstract

6S RNA is a highly abundant small RNA that regulates transcription through direct interaction with RNA polymerase. Here we show that 6S RNA directly inhibits transcription of pspF, which subsequently leads to inhibition of pspABCDE and pspG expression. Cells without 6S RNA are able to survive at elevated pH better than wild-type cells due to loss of 6S RNA-regulation of pspF. This 6S RNA-dependent phenotype is eliminated in pspF-null cells, indicating that 6S RNA effects are conferred through PspF. Similar growth phenotypes are seen when PspF levels are increased in a 6S RNA-independent manner, signifying that changes to pspF expression are sufficient. Changes in survival at elevated pH most likely result from altered expression of pspABCDE and/or pspG, both of which require PspF for transcription and are indirectly regulated by 6S RNA. 6S RNA provides another layer of regulation in response to high pH during stationary phase. We propose that the normal role of 6S RNA at elevated pH is to limit the extent of the psp response under conditions of nutrient deprivation, perhaps facilitating appropriate allocation of diminishing resources.6S RNA is a noncoding small RNA conserved in many bacteria (4,30,35). 6S RNA function remained a mystery for several decades until the discovery that this RNA makes specific interactions with RNA polymerase (RNAP) (32). Bacterial RNAP is a multisubunit enzyme consisting of a catalytically active core enzyme (␣ 2 , ␤, ␤Ј, ) that requires an additional specificity subunit () to form the holoenzyme (E) required for transcription initiation. 6S RNA forms stable, specific complexes with the housekeeping holoenzyme of RNAP (i.e., E 70 in Escherichia coli and E A in Bacillus subtilis) but does not interact with other holoenzymes, the core enzyme, or free subunits (30). E. coli 6S RNA makes direct contact with the 70 subunit within this 6S RNA-E 70 complex (32), suggesting that RNA binding specificity may be mediated through the 70 subunit in a manner analogous to that of DNA binding specificity. The 6S RNA is primarily double-stranded, with a large single-stranded region in the center. This highly conserved secondary structure is essential for the ability of 6S RNA to form stable complexes with E 70 (30). These data have suggested models in which 6S RNA functions as a direct competitor for promoter DNA interactions with RNAP (4,30,31,32).6S RNA interactions with E 70 result in altered transcription at specific promoters, either directly at several 70 -dependent promoters or indirectly at several S -dependent promoters (29). Observed transcriptional changes are greatest during late-stationary phase (Ն18 h of growth), a time when 6S RNA levels are maximal. Intriguingly, not all 70 -dependent promoters are sensitive to 6S RNA, even though most E 70 is complexed with 6S RNA during this time frame (29,32). Several promoters directly regulated by 6S RNA contain an extended Ϫ10 element, which is defined by a conserved TGN sequence immediately upstream of the Ϫ10 sequence element. Promoters with e...

Significant Bias against the ACA Triplet in the tmRNA Sequence of Escherichia coli K-12

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Legionella pneumophila 6S RNA optimizes intracellular multiplication

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