Abstract
RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the “stringent” response and regulator of cellular metabolism. The classical “long” RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the “stringent response” to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.
Highlights
Bacteria use several modified nucleotides as intracellular messengers, such as cAMP, c-di-GMP, c-di-AMP, cGMP, and ppGpp, with the latter being the first to be identified and one of the best studied [1,2]
Distribution of the RelA/SpoT Homologue (RSH) superfamily Iterative Hidden Markov Models (HMMs) searching of 1072 complete genome sequences and phylogenetic analyses enabled the identification of RSH sequences from genomes across the tree of life
RSH proteins are defined by the presence of a ppGpp synthetase (SYNTH, corresponding to the ‘‘RelA_SpoT’’ domain of Pfam) domain and/or a ppGpp hydrolase (HD) domain
Summary
Bacteria use several modified nucleotides as intracellular messengers, such as cAMP, c-di-GMP, c-di-AMP, cGMP, and ppGpp, with the latter being the first to be identified and one of the best studied [1,2]. The first type of stringent response to be investigated was the RelA-mediated response to amino acid limitation in E. coli [13,14] Under these conditions, accumulation of deacylated tRNA bound in the ribosomal A-site is sensed by RelA. Accumulation of deacylated tRNA bound in the ribosomal A-site is sensed by RelA This protein uses ATP and GDP (or GTP) to synthesise the alarmone nucleotide ppGpp (or pppGpp) in a synthetase ( referred to as SYNTH) domain [11]. We use the nomenclature Rel for the ancestral bifunctional RSH, and RelA and SpoT for the two homologs derived from Rel gene duplication in proteobacteria, as per [3], and refer to these three SYNTH and HD-containing proteins as ‘‘long RSHs’’. The precise function of these domains is unclear, but they are involved in mediating interand intra molecular interactions and regulating catalytic activity [12]
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