We have focused our studies on two types of post-transcriptional regulation of gene expression, regulation of mRNA degradation and translation by small regulatory RNAs and regulation of protein stability by energy-dependent proteases. RpoS, a central stress response regulator in Escherichia coli, is subject to both of these levels of control. Degradation of RpoS requires ClpXP protease, and RssB, a protein that presents RpoS to the protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. Deletion analysis of RssB indicates that sequences at the C-terminus are critical for proper release of the substrate to the protease; N-terminal sequences are required for interaction with the protease. RpoS translation is positively regulated by at least two small RNAs. The message upstream of the RpoS translation start folds into a hairpin that occludes ribosome binding and therefore translation. The small regulatory RNAs, DsrA and RprA, compete for the inhibitory stem of the hairpin, disrupting the secondary structure of the RpoS leader, allowing translation. The promoter of dsrAis regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation resides in a minimal promoter region of 36 base pairs; while many elements in this region contribute to the temperature regulation, the RNA polymerase interaction site at -10 is most critical. Our results suggest that changes in promoter structure may be mediating temperature regulation. RprA, identified as a multicopy suppressor of dsrAmutants, is regulated by the two component RcsC and RcsB regulators. These regulators also act to turn up capsular polysaccharide synthesis and to activate synthesis of a cell division protein; they are activated by cell surface stress. When they are activated, RpoS synthesis increases in an RprA-dependent fashion. It seems possible that this small RNA is particularly important during biofilm formation. A collaboration with Dr. Gisela Storz used our knowledge of the small RNAs described above to develop a strategy for finding novel small RNAs in E. coli, and more recently to find a particular class of small RNAs that bind an RNA chaperone, Hfq. The initial search resulted in the identification of 17 new small RNAs and six new, small ORFs. Different small RNAs are expressed under different growth conditions. Hfq, an RNA chaperone that is necessary for DsrA and RprA action, was found to bind to about 1/3 of the previously known small RNAs in E. coli, and potentially 20 new small RNAs were identified by their binding to Hfq. Hfq stabilizes the small RNAs and helps them pair to mRNA targets. Once paired to the targets, they can either stimulate translation (as for DsrA and RprA) or cause message degradation. One novel small RNA, RyhB, has been investigated in some detail. We find that it is repressed by the Fur, iron-dependent repressor, and is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets multiple mRNAs for degradation. The target mRNAs encode either iron storage proteins (ferritins) or iron-containing but non-essential metabolic proteins. Therefore, this small RNA, which is also found in Vibrio, Salmonella, , Klebsiella, , and Yersinia, , reprograms iron use in the cells and may be an important component of virulence for some pathogens. When the target message is degraded, so is RyhB; both molecules require RNaseE for degradation. This coupled degradation appears to be true for other small RNAs as well. The other novel small RNAs are likely to represent equally interesting new regulatory pathways. In addition, the information gained from our genome-wide search for small RNAs in E. coli is forming the basis for extending the search for small RNAs to other bacterial species.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC008714-26
Application #
6950495
Study Section
(LMB)
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
De Lay, Nicholas; Gottesman, Susan (2009) The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior. J Bacteriol 191:461-76
Bougdour, Alexandre; Cunning, Christofer; Baptiste, Patrick Jean et al. (2008) Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors. Mol Microbiol 68:298-313
Bougdour, Alexandre; Gottesman, Susan (2007) ppGpp regulation of RpoS degradation via anti-adaptor protein IraP. Proc Natl Acad Sci U S A 104:12896-901
Majdalani, Nadim; Gottesman, Susan (2007) Genetic dissection of signaling through the Rcs phosphorelay. Methods Enzymol 423:349-62
Thompson, Karl M; Rhodius, Virgil A; Gottesman, Susan (2007) SigmaE regulates and is regulated by a small RNA in Escherichia coli. J Bacteriol 189:4243-56
Vanderpool, Carin K; Gottesman, Susan (2007) The novel transcription factor SgrR coordinates the response to glucose-phosphate stress. J Bacteriol 189:2238-48
Ranquet, Caroline; Gottesman, Susan (2007) Translational regulation of the Escherichia coli stress factor RpoS: a role for SsrA and Lon. J Bacteriol 189:4872-9
Bougdour, Alexandre; Wickner, Sue; Gottesman, Susan (2006) Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli. Genes Dev 20:884-97
Zhou, YanNing; Gottesman, Susan (2006) Modes of regulation of RpoS by H-NS. J Bacteriol 188:7022-5
Tu, Xuanlin; Latifi, Tammy; Bougdour, Alexandre et al. (2006) The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica. Proc Natl Acad Sci U S A 103:13503-8

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