The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus

Abstract

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.

Endoribonuclease YbeY Is Essential for RNA Processing and Virulence in Pseudomonas aeruginosa

“…Studies in E. coli demonstrated its essentiality in the biogenesis of the 50S ribosomal subunit (56). In bacteria, the colocalization of ybeZ and ybeY in an operon is highly conserved (36)(37)(38). Here, we demonstrated the interaction between YbeY and YbeZ in P. aeruginosa and found that mutation of ybeZ resulted in similar phenotypes as the ybeY mutant.…”

“…However, the mechanisms by which YbeY affects bacterial virulence and stress response remain unclear. Among bacterial species, including P. aeruginosa, E. coli, and Staphylococcus aureus, the ybeY gene is colocalized with a ybeZ gene in the same operon (36)(37)(38). In E. coli, YbeY has been found to interact with YbeZ (37), indicating a functional connection between the two proteins.…”

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes

“…Finally, we would like to draw the attention of the research community working on long RSH enzymes to technical aspects of protein purification. It is common to purify Rel/RelA for biochemical experiments using a single-step purification [for example (Wood et al, 2019)]. However, both RelA (Turnbull et al, 2019) and Rel have a strong tendency for RNA contamination and multiple additional steps are necessary to remove this contaminant.…”

“…Without extra steps to remove RNA contamination, single-step preparations are likely to be heavily contaminated with ribosomal particles (Figure 6), which is likely to interfere with the estimation of the oligomerization state of Rel, since the RNA-bound protein elutes much earlier than the RNA-free fraction (Figures 5B,C). This contamination may explain the surprising observation that the addition of Ni-NTA purified S. aureus Rel inhibits the 50S assembly factor DEAD-box RNA helicase CshA (Wood et al, 2019). The unlabeled contaminating ribosomal particles could potentially be recognized by CshA, thus acting as a competitor in the helicase assay that uses a synthetic Cy3-labeled RNA duplex as a substrate.…”

“…The unlabeled contaminating ribosomal particles could potentially be recognized by CshA, thus acting as a competitor in the helicase assay that uses a synthetic Cy3-labeled RNA duplex as a substrate. It is also plausible that formation of stable complexes of Rel with ribosomes in live E. coli could generate false-positive signals in bacterial two-hybrid assays, accounting for the observed protein-protein interaction between S. aureus Rel and the ribosome assembly factors Era and CshA (Wood et al, 2019).…”