Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain.
The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA(+) cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752-5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.
Gropp
M
M
Department of Cellular Biochemistry, The Hebrew University-Hadassah Medical School, Jerusalem 90101, Israel.
Strausz
Y
Y
Gross
M
M
Glaser
G
G
D037361:Adenylate Cyclase Toxin; D000596:Amino Acids / Q000172:deficiency; D001426:Bacterial Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006158:Guanosine Pentaphosphate / Q000378:metabolism; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D037361:Adenylate Cyclase Toxin; D000596:Amino Acids; D001426:Bacterial Proteins; D011498:Protein Precursors; D006159:Guanosine Tetraphosphate; D006158:Guanosine Pentaphosphate; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases