Intramolecular interactions dominate the autoregulation ofEscherichia colistringent factor RelA

Abstract

AbstractAmino acid starvation inEscherichia coliactivates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as ‘starved’ ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act eitherin cis(inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) orin trans(CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains ofE. coliRelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulationviainhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibitionviadimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likelyviadirect interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.