Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions

BACKGROUND: The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. RESULTS: NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. CONCLUSIONS: The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0502-9) contains supplementary material, which is available to authorized users

Not Available

Not AvailableNot AvailableNot Availabl

Not Available

Not AvailableAbstractNot Availabl

Not Available

Not AvailableNot AvailableNot Availabl

Not Available

Not AvailableNot AvailableNot Availabl

Not Available

Not AvailableNot AvailableNot Availabl