We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia coli as a model system. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to regulate RpoS activity as well as degradation. Extracts overproducing RssB but not those devoid of RssB are able to stimulate the ClpXP-dependent degradation of RpoS, allowing the development of an assay for purification, to lead to detailed study of the factors that stimulate degradation. RpoS translation is regulated by DsrA, a small stable 85 nucleotide RNA. Mutational analysis indicates that a stem-loop at the 5' end of the RNA is necessary for regulation of RpoS, and shows homology to a region of the untranslated leader of RpoS that has been implicated in translational regulation. This suggests that pairing between the DsrA RNA and the RpoS leader opens up the leader structure, allowing translation. The second activity of DsrA, overcoming the silencing associated with the nucleoid associated HNS protein, is independent of the first stem-loop, demonstrating that this RNA has two distinct activities. We have used cI-HNS hybrid proteins, in which immunity to bacteriophage lambda depends on oligomerization between the HNS domains, to demonstrate that anti-silencing works by disrupting HNS oligomerization. DsrA and other small RNAs are gaining increasing recognition as novel regulatory molecules. In studies on the degradation of the lambda Xis protein, we find that it is subject to degradation by two distinct E. coli ATP-dependent proteases, Lon and FtsH. Rapid degradation of Xis helps insure rapid and stable lysogenization by lambda. In mutagenesis studies of the Lon protease, we find that deletions and point mutations in the C-terminal domain that remove or inactivate the proteolytic active site are still able to partially complement lon mutants when overproduced. This appears to be because the substrate binding domain is retained in the N-terminus of the deleted proteins, and binding of substrate is sufficient to interfere with substrate function, mimicking the effect of degradation. This will allow the analysis of substrate recognition and binding by the Lon protease to be studied independently of protein degradation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC008714-21
Application #
6100921
Study Section
Special Emphasis Panel (LMB)
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
1998
Total Cost
Indirect Cost
Name
National Cancer Institute Division of 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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