Escherichia coli RelA Regulation via Its C-Terminal Domain

Abstract

One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli, RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564-744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell.

Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint

“…While full-length A. baumannii SpoT, E. coli RelA and B. subtilis Rel 25 , as well as M. tuberculosis Rel 40 all behave as monomers in our hands, previous results indicate the possibility of long-RSH regulation via oligomerization 22 , 28 , 43 . However, the oligomerization model is largely supported by experiments with truncated protein variants, and these engineered RSH could potentially form contacts in trans that are naturally formed in cis in the full-length factor.…”

“…It was shown earlier that Rel and RelA are prone to dimerization, a potential factor in their regulation 22,[27][28][29] . This idea is subject of debate, with genetics 30 and mass photometry 25 suggesting that dimerization is unlikely to take place at physiological concentrations.…”

New Chemotypes for the Inhibition of (p)ppGpp Synthesis in the Quest for New Antimicrobial Compounds

“…“Short” RSH proteins harbor either only the synthetase (SAS, small alarmone synthetases) or hydrolase (SAH, small alarmone hydrolases) domain, respectively. “Long” RSH proteins contain both catalytic domains and a C-terminal regulatory domain (CTD) that activates alarmone synthesis by promoting Rel oligomerization [ 21 ] and/or upon binding to stalled ribosomes (i.e., ribosomes bound to uncharged tRNAs during aminoacid starvation) [ 22 , 23 ] or favors alarmone hydrolysis by directly inhibiting the synthetase site [ 24 ]. In addition, reciprocal regulation of the two catalytic domains has been postulated with mechanisms that vary among species [ 25 , 26 , 27 , 28 ].…”

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis

“…coli and other β- and γ-Proteobacteria. During unstressed growth, it is proposed that the synthetase function of RelA is kept inactive through the formation of RelA homodimers that form via intermolecular disulfide bonds between adjacent CTDs [ 21 – 23 ]. However, this regulatory mechanism has been challenged by work showing that the expression of the CTD or full-length RelA does not compromise the ability of E .…”