Escherichia coli RelA Regulation via Its C-Terminal Domain.

Ilana Kaspy,Gad Glaser

doi:10.3389/fmicb.2020.572419

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.

Highlights

To survive, bacteria must be able to respond to changes in their environment
Cross-linking experiments and mass spectrometry (MS)-MS analysis revealed the ability of the native RelA and YG4 to form dimers via the formation of S-S bonds between Cys-638 both between the full length proteins and between YG4 fragments (Figure 4), which we believe exist in vivo
This was recently shown in Rel protein from B. subtilis, where Rel is in an oligomeric state in the cytosol during normal growth conditions, but upon accumulation of uncharged tRNA the dimer dissociates by interaction with the C-terminal domain (CTD) and together binds to a cognate ribosome (Pausch et al, 2020)

Summary

Introduction

Bacteria must be able to respond to changes in their environment. Depriving Escherichia coli of one or more amino acids (AAs) triggers the stringent response (Stent and Brenner, 1961; Cashel, 1969; Cashel and Gallant, 1969; Kaspy et al, 2013). In extracts of normally growing cells, RelA is associated with a small fraction (about 1%) of the ribosomes (Pedersen and Kjeldgaard, 1977). Both physically and functionally, E.coli RelA includes two distinct domains: the N-terminal domain [NTD; amino acids (AAs) 1-455], which is responsible for (p)ppGpp synthesis and the C-terminal domain (CTD; AAs 405–744), which is responsible for regulating RelA activity (Metzger et al, 1989; Schreiber et al, 1991; Gropp et al, 2001). Much work has been devoted to trying to decipher the regulatory mechanism of the Rel protein family (Wendrich et al, 2002; Agirrezabala et al, 2013; Turnbull et al, 2019; Takada et al, 2020)

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Results

Conclusion