Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

Abstract

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.

Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS

“…In E. coli, ppGpp enhances the transcription of rpoS (8,17,41) and many rpoS-dependent genes (28). Therefore, we examined the role of relA in the expression of hapA.…”

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

“…RelA associates with ribosomes, and synthetase activity is triggered upon an accumulation of uncharged tRNA sensed by the ribosome during amino acid depletion (10)(11)(12)(13). The synthetase activity of SpoT, on the other hand, is induced by other stresses such as fatty acid depletion (1).…”

A Truncated Form of SpoT, Including the ACT Domain, Inhibits the Production of Cyclic Lipopeptide Arthrofactin, and Is Associated with Moderate Elevation of Guanosine 3′,5′-Bispyrophosphate Level inPseudomonassp. MIS38

“…Dissection of E. coli RelA described the feature of the C-terminal domain that senses amino acid starvation, possibly via interaction with the ribosome. 23,24) The dualfunction of SpoT, possessing both ppGpp hydrolase and pppGpp synthetase activities, is similar to those of the RSH proteins in most Grampositive bacteria. The crystal structure of Rel seq from Streptococcus equisimilis suggests a model for the catalysis of RSH, in which a hinge-like movement of the C-terminal region alternatively blocks the separate active sites in the N-terminal region responsible for the hydrolysis and synthesis of (p)ppGpp.…”