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 Rel Sau , with Rel Sau 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.
Highlights
Ribosomes are macromolecular machines responsible for the synthesis of proteins in all living cells
We examine the function of Era, a GTPase enzyme involved in 30S ribosomal subunit biogenesis in the important human pathogen S. aureus
We uncover that Era is not an essential enzyme in S. aureus, as it is in many other species, but is important for correct ribosome assembly
Summary
Ribosomes are macromolecular machines responsible for the synthesis of proteins in all living cells. Assembly of the ribosome is a tightly regulated process, with correct maturation requiring the help of assembly cofactors, one class of which are the P-loop ribosome-associated GTPases (RA-GTPase) These enzymes are widely conserved across all domains of life and act as molecular switches, cycling between inactive GDP-bound, and active, effector-binding GTP-bound states. The protein after which this family is named, is highly conserved throughout the bacterial kingdom, is missing in Chlamydia and mycobacterial species [1] This GTPase is essential in E. coli [2,3,4,5], Salmonella Typhimurium [6] and in some strains of Bacillus subtilis, where deletion mutants of B. subtilis strains IS75 and CRK6000 were unobtainable [7, 8], an era knockout was created in strain BR151 [7]. While important for ribosomal maturation, additional phenotypic defects associated with depleted cellular levels of Era include cell cycle control and chromosome segregation, as well as carbon and nitrogen metabolism [10,11,12]
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