The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus

Abstract

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

Hibernation-Promoting Factor Sequesters Staphylococcus aureus Ribosomes to Antagonize RNase R-Mediated Nucleolytic Degradation

“…We propose a model by which hibernating ribosomes (HPF-bound 70S and 100S) serve as a reservoir to preserve unused ribosomes. When a demand for translation increases, e.g., upon exit from dormancy and in response to a specific stressor, ribosome hibernation is reversed via dissociation and recycling of ribosomes split by either the RRF/EF-G disassembly pathway ( 24 ) or the heat-induced HflX-mediated pathway ( 25 ), allowing initiation of new translation ( Fig. 8A ).…”

“…There is no contact between the RMF and the short form HPF in E. coli ; instead, RMF allosterically induces a "back-to-back" joining of 70S monomers at the 30S-30S interface to form the 90S dimer, followed by the short HPF-mediated stabilization of the 100S ribosome and translational inhibition resembling that of a CTD-HPF ( 15 , 23 ). To reactivate hibernating ribosomes for translation and exit from dormancy, S. aureus 100S ribosomes are disassembled into recyclable 30S and 50S subunits or 70S complexes by either the RRF/EF-G pathway or through the heat-induced GTPase HflX ( 24 , 25 ).…”

Hibernation as a Stage of Ribosome Functioning

“…We assume that hibernation/silencing proteins can bind to free ribosomal subunits and vacant 70S ribo somes, forming the 50S RsfS [31], 30S YfiA [49], 70S YfiA [49], 100S l HPF or 100S RMF/HPF [69] com plexes. Later on, these complexes can be disassembled to the 30S and 50S subunits by the cooperative action of RRF and EF G or by HflX (heat shock GTPase) in the case of heat shock [70]. However, if the hibernation pro teins RMF, HPF, YfiA, and RsfS are present in relatively large amounts, the ribosomal subunits can again form inactive complexes (Fig.…”

MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy

“…To resume translation ribosome hibernation is reversed by 100S dissociation. Recently, it was shown that in S. aureus ribosome recycling factor (Rrf, also known as Frr) and elongation factor G (EF-G, also known as FusA) synergistically split 100S dimers and, in certain conditions, this process may include activity of HflX GTPase ( 52 ). Notably, we observed that MazF decreased levels of rrf ( Supplementary Figure S14A ).…”