DsrA is a small, stable, untranslated RNA that is conserved between Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium. DsrA RNA regulates two different global regulatory networks. 1) DsrA antagonizes transcriptional repression by the nucleoid-associated protein H-NS. Overexpression of DsrA mimics an HNS phenotype without modulating H-NS levels in the cell or by antagonizing the DNA binding ability of H-NS in vivo. H-NS is an important regulator of virulence factors and other genes in E. coli and related bacteria. In Shigella spp. and enteroinvasive E. coli, H-NS (VirR) represses the expression of virulence genes at low temperature and low osmolarity. In Salmonella typhimurium, H-NS negatively regulates the spv virulence locus and mutations in H-NS lead to decreased virulence in mice. Both plasmid and phage encoded Shiga-like toxin II are also regulated by H-NS. Numerous other genes not directly associated with virulence are also regulated by DsrA/H-NS. 2) The chromosomal copy of DsrA is essential for the high level translation of the alternate sigma factor, RpoS during exponential growth at low temperature (less than 30 degrees C). This DsrA-dependent accumulation of RpoS is sufficient to cause increased expression of RpoS-dependent transcriptional fusions normally expressed only in stationary phase at 37 degrees C. RpoS is important for the virulence of E. coli and S. typhimurium as well as their survival and adaptation to numerous environmental stresses. A mutational analysis of DsrA suggests that these two regulatory activities are separable and correlate with different portions of the DsrA RNA molecule. Since DsrA regulates both networks it is likely that DsrA itself plays a role in the virulence of these organisms by modulating their ability to adapt to stressful environments. The long term objective of this study is to determine how DsrA, as an RNA, regulates gene expression and the role of DsrA in the virulence of E. coli and S. typhimurium. The hypothesis to be tested is that DsrA, either alone or with the coregulator HF-I, binds to H-NS and interferes with it oligomerization on specific DNA target sites.
The specific aims are: 1) To characterize the interaction of DsrA and H-NS. DsrA/H-NS interactions will be analyzed in vivo using H-NS-cI protein hybrids, these hybrids will also be used in a mutational analysis of H-NS to define a site(s) of interaction between DsrA and H-NS. Purified DsrA and H-NS will be used to characterize potential interactions in vitro. To determine whether H-NS and DsrA RNA directly interact, the in vivo dimethylsulfate protection pattern of DsrA RNA will be compared in both hns+ and hns strain backgrounds. 2) To characterize the role of HF-I (hfq) an RNA binding protein that is essential for DsrA activity in vivo. HF-I will be purified and DsrA- specific hfq mutants isolated. To determine whether HF-I binds to and/or alters the structure of the DsrA RNA, the in vivo dimethylsulfate protection pattern of DsrA RNA will be compared in both hfq+ and hfq strain backgrounds. Similar experiments using in vitro synthesized DsrA and purified HF-I will also be done. 3) To compare the effect of DsrA and H-NS on DNA structure in vivo. In vivo footprinting of multicopy plasmid genes regulated by H-NS/DsrA will be used to compare the effects of hns-mutations and overexpression of DsrA on DNA structure.
| Brescia, Cristin C; Kaw, Meenakshi K; Sledjeski, Darren D (2004) The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo. J Mol Biol 339:505-14 |
| Brescia, Cristin C; Mikulecky, Peter J; Feig, Andrew L et al. (2003) Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA 9:33-43 |
